- Email: [email protected]
Epicardium and Myocardium Originate From a Common Cardiogenic Precursor Pool
REVIEW ARTICLES Epicardium and Myocardium Originate From a Common Cardiogenic Precursor Pool Bram van Wijk and Maurice van den Hoff⁎
During development, the epicardium, an epithelial layer that covers the heart, gives rise to a large portion of the nonmyocardial cells present in the heart. The epicardium arises from a structure, called the proepicardium, which forms at the inflow of the developing heart. By epithelial-tomesenchymal transformation, mesenchymal cells are formed that will subsequently populate the stroma of the proepicardium and the subepicardium. Based on labeling analysis, the proepicardium and part of the myocardium have been shown to be derived from a common cardiogenic precursor population. In this review, we will discuss the common cardiogenic origin of proepicardial and myocardial cells, the underlying processes and factors that play a role in the separation of the lineages, and their potential role in cardiac regenerative approaches. (Trends Cardiovasc Med 2010;20:1–7) n 2010, Elsevier Inc.
Introduction
Given the limited regenerative capacity of the adult heart, cardiac damage frequently progresses to congestive heart failure. Attempts to stimulate cardiac repair have seen large efforts being made to identify and isolate cardiac progenitors. Several lines of evidence suggest that the epicardium, the nonmyocardial epithelium covering the myocardium, could conceivably play a role in cardiac regeneration. In zebrafish, reac-
Bram van Wijk and Maurice van den Hoff are at the Heart Failure Research Center, Academic Medical Center, 1105AZ Amsterdam, The Netherlands. ⁎ Address correspondence to: M.J.B. van den Hoff, PhD, Academic Medical Center, Department of Anatomy, Embryology & Physiology, Meibergdreef 15, 1105AZ Amsterdam, The Netherlands. Tel.: (+31) 205665415; fax: (+31) 206976177; e-mail: m.j.vandenhoff@ amc.uva.nl. © 2010, Elsevier Inc. All rights reserved. 1050-1738/$-see front matter
TCM Vol. 20, No. 1, 2010
tivation of the epicardium plays a critical role in the regeneration of injured myocardium (Lepilina et al. 2006). Although the mammalian heart possesses little regenerative capacity in contrast to the heart of zebrafish, its epicardium has also been suggested to be involved in myocardial regeneration (Di Meglio et al. 2009; Limana et al. 2007, 2009; Winter et al. 2007). During development, the epicardium arises from a cauliflower-like structure called the proepicardium. Around the third day of development (Hamburger– Hamilton stage 16) in chicken, 9.5 days of development in mouse, and ≈3.5 weeks (Carnegie stage 10) of human development, the proepicardium arises from splanchnic mesoderm that covers the vitelline veins at the inflow of the developing heart (Manner et al. 2001). Until this stage, the heart lies “naked” within the pericardial cavity. The proepicardium protrudes into the pericardial cavity and contacts the myocardium of the atrioventricular canal. Subsequently, epicardial cells migrate over the heart
tube to cover the myocardium. Within the proepicardium and later also in the epicardium that covers the heart tube, a subset of the epithelial cells undergoes epithelial-to-mesenchymal transformation (EMT) (Wessels and Perez-Pomares 2004). These mesenchymal cells provide a large portion of the nonmyocardial cells of the heart, for instance, cardiac fibroblasts, coronary smooth muscle cells, and coronary endothelial cells. Although still an issue of debate (Christoffels et al. 2009), recent genetic labeling studies suggest that cells derived from the epicardium might contribute to the myocardial lineage, as well (Cai et al. 2008; Zhou et al. 2008a). The wide variety of cells the proepicardium contributes to the heart (Cai et al. 2008; Gittenberger-de Groot et al. 1998; Manner 1999; Perez-Pomares et al. 1997; Wessels and Perez-Pomares, 2004; Zhou et al. 2008a), coupled with the fact that the (pro)epicardium and myocardium share a common cardiogenic precursor (Van Wijk et al. 2009; Zhou et al. 2008a,b), makes epicardial cells a potential source of cardiac progenitor cells and as such may serve a potential source of cells for regenerative approaches. In this review, we will discuss the common origin of epicardial and myocardial cells, the processes and factors that play a role in the separation of myocardial and nonmyocardial lineages, and the potential role of epicardial-derived cells as progenitor cells.
Common Origin of Epicardial and Myocardial Cells
Classic Lineage Tracing Analyses The heart is formed from cells of the splanchnic mesoderm. The bilateral zones of the splanchnic mesoderm, which will derive the heart, are referred to as the heart-forming regions. Within these regions, cells have been subdivided into two developmental fields: the first and second heart fields (Buckingham et al. 2005). This distinction should be considered a spatiotemporal separation
1
of cells within a greater heart-forming region (Rochais et al. 2009). The lateral cells of this region, the first heart field, are the first to exit the cell cycle, subsequently differentiating into myocardium and giving rise to the primary heart tube. The cells located medially and caudally within the heart-forming region are referred to as the second heart field (Figure 1). These cells continue to proliferate in an undifferentiated state after the formation of the linear heart tube and are gradually added to both the inflow and outflow regions of the heart (Van Den Berg et al. 2009) as it loops. At the inflow of the heart, these progenitors contribute both myocardial and nonmyocardial cells. The nonmyocardial component of the heart is largely derived from cells of the proepicardium that develops at the inflow of the heart. In the cardiac progenitor pool, present upstream of the heart tube before the induction of the proepicardium, a lineage decision takes place, defining cells that will differentiate into cardiomyocytes and be added to the venous pole of the heart or cells that will differentiate into the proepicardial lineage (Van Wijk et al. 2009). An in-depth study of the various cell types contributing to the inflow of the heart and the proepicardium has long been lacking. Classic lineage analyses, with the use of vital dyes, radioactive deposits, or retroviruses, were hampered because the venous pole of the heart is difficult to approach in the living embryo owing to technical limitations. Only recently have we been able to demonstrate the contribution of the second heart field to both the inflow myocardium and the proepicardium with the use of a vital dye labeling (Van Wijk et al. 2009). In this analysis, a small group of splanchnic mesodermal cells, expressing the T-box transcription factor 18 (Tbx18) at the inflow region of the heart, was labeled 1 day before the initiation of proepicardium formation (stage 11). At the time of labeling, these cells neither were positive for myocardial markers, like cardiac troponin I or myosin heavy chain isoforms, nor showed morphologic characteristics of proepicardium formation. Twenty-four hours later, the label was detected both in myocardial cells of the inflow tract and in proepicardial cells. Because, in this experiment, a small group of cells was labeled rather
2
than a single cell, it cannot be excluded that the labeled population comprises a mixed population instead of bipotential progenitor cells. However, this would seem unlikely, as gene expression analysis of the mesoderm at the stage of labeling, that is, before formation of the proepicardium, displays no heterogeneity in gene expression. Earlier cell tracing studies in which (1) quail proepicardia were transplanted into chicken embryos (Manner 1999; Perez-Pomares et al. 1997), (2) adenovirus-labeled chicken proepicardia were transplanted into chicken acceptors (Dettman et al. 1998), and (3) proepicardia were retrovirus-labeled in ovo (Mikawa and Fischman, 1992) were unable to demonstrate proepicardial contribution to the myocardium. Although cell tracing analysis revealed no contribution of proepicardial cells to the myocardium, proepicardial cells have been shown to differentiate into heart muscle cells in vitro (Kruithof et al. 2006; Schlueter et al. 2006; Van Wijk et al. 2009). The two most important differences between the recent labeling experiments and the earlier analyses are the developmental stage at which the cells were labeled and the techniques used to label the proepicardia. In the recent experiment, the mesoderm upstream of the forming heart tube was labeled before the induction and formation of the proepicardium at stage 11, whereas in the earlier experiments, the proepicardium was labeled later, at stage 16 to 17. Second, in the studies in which proepicardia were transplanted, the donor tissue was not in direct contact with the inflow of the recipient embryo. Consequently, the physical barrier, that is, the pericardial cavity, prevented the transplanted cells from contributing to the inflow myocardium. The expression of Tbx18 first becomes evident in the splanchnic layer immediately upstream of the heart tube at stage 11 (Haenig and Kispert 2004; Van Wijk et al. 2009). One day later during development, Tbx18 expression can be observed in distinct regions at the inflow of the heart, being (1) myocardial cells just cranial to the base of the proepicardium (Kruithof et al. 2006; Van Wijk et al. 2009); (2) sinus horn myocardium, that is, the myocardial sleeves covering the cardinal veins (Christoffels et al. 2006); and (3) proepicardial cells (Haenig and
Kispert 2004; Kraus et al. 2001). Histologic analysis of the proepicardium has shown that the proepicardial villi are sheathed in epithelial cells and filled with mesenchymal cells and extracellular matrix. Because of the presence of genes involved in EMT, like Wt1 and Snail, in the proepicardium (Carmona et al. 2001), it is likely that during proepicardium formation epithelial cells transform into mesenchymal cells that invade the proepicardial matrix. Within the proepicardium, several genes have been reported to be heterogeneously expressed, for example, P-Erk (Van Wijk et al. 2009), FGF2 (Kruithof et al. 2006), Flk1 (Cai et al. 2008), PAR3, and ZO1 (Hirose et al. 2006), indicating that the proepicardium is a heterogeneous population of cells. Taken together, a homogeneous, Tbx18-expressing mesodermal cell population upstream of the inflow of the heart, which covers the vitelline veins at stage 11, gives rise to proepicardial and inflow myocardial cells. Within the proepicardium, epithelial cells transform into mesenchymal cells that will subsequently invade the proepicardial matrix. Genetic Lineage Analyses The development of a two-component mouse system, in which one mouse harbors a conditional reporter gene, like lacZ or GFP, and the other mouse expresses Cre (Causes recombination), has enabled genetic lineage tracing analyses to be carried out (Soriano 1999). The last couple of years have provided a variety of different Cre lines that have been used to analyze the development of the inflow of the heart. The Nkx2.5IRES-Cre, in which an IRESCre cassette is inserted into the 3′UTR of the Nkx2.5 gene (Stanley et al. 2002), showed robust reporter gene expression in all myocardial cells and the proepicardium, but not in sinus horn myocardium (Christoffels et al. 2006; Stanley et al. 2002). Similar results were obtained with the use of an Nkx2.5Cre line (Zhou et al. 2008b) or an Isl1Cre line (Cai et al. 2003; Ma et al. 2008), in which Cre is knocked in at the translation start sites. In situ hybridization and immunohistochemical analyses of the inflow tract have shown that neither the proepicardium nor the sinus horn myocardium expresses Nkx2.5 or Isl1 (Christoffels et
TCM Vol. 20, No. 1, 2010
Figure 1. Initial heart formation. Panels A, B, and C spatially illustrate early heart formation in chicken from the ventral side. A, The heartforming regions (HFR) are indicated in gray (primary heart field, Nkx2.5 lineage) and blue (second heart field, Isl1 lineage). The dotted red line indicates the area of the HFR that will express Wt1 and the transcription factor Tbx18. B, The bilateral HFR adjoin in the midline by folding of the embryonic disc (hatched arrows). The red line indicates the area of the HFR that expresses Wt1 and Tbx18. C, Progressive folding lengthens the heart tube in caudal direction, forms the foregut, and establishes the pericardial back wall.
al. 2006; Ma et al. 2008; Zhou et al. 2008b). These findings reveal that cells of the proepicardium and sinus horn myocardium are derived from progenitors that have at some point expressed Nkx2.5 and Isl1 during development, though not necessarily at the same time. This suggest that both proepicardium and sinus horn progenitors are derived from the second heart field, but turn off isl1 earlier, and do not activate nkx2.5 like other second heart field progenitors. However, the relation between the proepicardial and sinus horn progenitors is currently unclear. Indeed, sinus horn myocardium is formed subsequent to the formation of the proepicardium, that is, from E9.5 onward, and, in contrast to epicardium and all other myocardium, is unrecombined in Nkx2.5IRES-Cre;R26R embryos, suggesting a much briefer period of Nkx2.5 expression in these progenitors compared with the proepicardial progenitors
TCM Vol. 20, No. 1, 2010
(Christoffels et al. 2006). Consequently, the myocardium located directly cranial to the proepicardium is different from sinus horn myocardium. In the neonatal heart, the myocardium directly cranial to the proepicardium finds its destiny in the myocardium of the sinus node and right venous valve (Wiese et al. 2009). Because the insertion of Cre in the Nkx2.5Cre and Isl1Cre lines disrupts expression of functional Nkx2.5 and Isl1 protein, respectively, their role in proepicardial development could be tested. Homozygous disruption of Nkx2.5 or Isl1 results in growth retardation and cardiac malformation. In Nkx2.5Cre/Cre mice, the proepicardium fails to develop, whereas in Isl1Cre/Cre, the proepicardium remains underdeveloped (Zhou et al. 2008b). These findings suggest that Nkx2.5, although not expressed in the proepicardium, is required for its formation, this not being the case for Isl1. Isl1 seems to be required for proper expan-
sion of the cardiac progenitor pool (Prall et al. 2007) and consequently for providing sufficient progenitors for proepicardial development. Analysis of Tbx18Cre mice revealed recombination in the forming epicardium and adult heart (Cai et al. 2008). Wilms tumor 1 (Wt1)-Cre, another Cre line of importance in (pro)epicardial development, demonstrated a similar pattern of recombination, that is, in the epicardium and in cells of the adult heart (Zhou et al. 2008a) (details of both Cre lines will be discussed later in this review). The contribution of the Tbx18Cre and Wt1Cre lineage to the inflow myocardium has not been addressed in these studies. Wt1-expressing proepicardial cells were shown to be derived from cells that were found to belong to both the Nkx2-5 and Isl1 lineages, even though Wt1 protein was never found to be coexpressed with Isl1 nor Nkx2-5 proteins (Zhou et al. 2008a).
3
Taken together, labeling studies of Tbx18-expressing progenitors (Van Wijk et al. 2009), together with lineage analyses with the use of Isl1- and Nkx2-5-Cre lines (Ma et al. 2008; Zhou et al. 2008b), have identified a common origin of myocardial and (pro)epicardial cells.
Regulation of the Separation of Myocardial and Nonmyocardial Cells
The earliest markers that identify the myocardial and proepicardial progenitor population are Tbx18 and Wt1. Expression of Tbx18 and Wt1 starts just before E8.0 in mouse (Kraus et al. 2001; Zhou et al. 2008a) and stage 11 in chicken (Haenig and Kispert, 2004; Van Wijk et al. 2009). Both in mouse (E9.5) and chicken (stage 16), the proepicardium starts to become morphologically evident as villous structures protruding into the pericardial cavity (Schulte et al. 2007). Proepicardial villi appear on both the left and right vitelline veins. At the right side, the villi expand and form the proepicardium, whereas at the left side, in chicken, the villi undergo apoptosis and disappear. The pathways regulating this left–right asymmetry are beyond the scope of this review and have been discussed elsewhere (Schlueter and Brand, 2009). By contrast, in mouse, the villi expand and form a proepicardium that is located medially (Schulte et al. 2007). The factors that induce expression of Tbx18 and Wt1 are presently unknown. However, xenotransplantion of stage 17 quail liver buds to a caudal location in chicken of various developmental stages showed induction of Tbx18 and/or Wt1 expression in the adjacent mesoderm from stage 12 onward, indicating that secreted factors derived from the hepatic endoderm induce the expression of Tbx18 and Wt1. Similarly transplanting lung buds, as a control, did not have this effect, hinting that the forming hepatic endoderm is the probable source of factors that induce expression of Tbx18 and Wt1 in the splanchnic mesoderm. Because lung buds produce Bmp and Fgf, these growth factors are unlikely candidates to induce the expression of Tbx18 and Wt1 (Ishii et al. 2007; Van Wijk et al. 2009). Although Tbx18 marks the progenitor population of proepicardial and inflow myocardial cells, its expression is not essential for the formation of the proepicardium because func-
4
tional disruption of Tbx18 does not result in aberrant proepicardial development (Christoffels et al. 2006). Homozygous deletion of Wt1, on the other hand, severely impairs (pro)epicardial development (Moore et al. 1999). Wt1's involvement in EMT suggests that EMT plays an important role in the formation of proepicardial cells (Carmona et al. 2001). Besides EMT, the regulation of cell polarity also seems to be involved in proepicardium formation, as deletion of the mammalian homologue of the Caenorhabditis elegans polarity protein, Par3, results in defective (pro)epicardial development (Hirose et al. 2006). The factors that regulate the division of the progenitor population into myocardium on the one hand and proepicardium on the other are largely unknown. Although Bmp and Fgf do not induce the expression of Tbx18 or Wt1 in this progenitor population (Ishii et al. 2007), they do play a role in the separation of myocardial and nonmyocardial cells from this population (Van Wijk et al. 2009). Exposing embryos to Bmp2, Fgf2, and/or the Mek1 inhibitor U0126 has shown that Bmp2 and Fgf2 signaling pathways cooperate in the separation and differentiation of the Tbx18-positive progenitor pool into cardiomyocytes and proepicardial cells. Smad-mediated Bmp-signaling directs the progenitors into the myocardial lineage. Erk-mediated Fgf2 signaling, on the one hand, directs the progenitors into the proepicardial lineage and blunts the differentiation of the progenitors into the myocardial lineage (Van Wijk et al. 2009). The inhibition of myocardial differentiation of the progenitors by Fgf2 was found to be the result of disrupting Smad-mediated Bmp-signaling by phosphorylating receptor Smad, such that it cannot interact with common Smad4 and thus accumulates in the cytoplasm. As a consequence, the initiated Bmp signal is not conveyed to the nucleus, thereby disrupting the Bmp response (Figure 2) (Van Wijk et al. 2009). Taken together, these data indicate that (an) unknown signal(s) secreted from the hepatic endoderm induces Tbx18 and Wt1 expression in the precardiac progenitor population. Separation of this progenitor population into the myocardial and epicardial lineage is regulated by the cooperative interaction
between Bmp and Fgf-signaling (Ishii et al. 2007; Schlueter et al. 2006; Van Wijk et al. 2009).
Proepicardial Contribution to the Heart
Xenotransplantation studies with the use of the quail–chick chimera technique as well as cell fate analyses with the use of retroviral and adenoviral cell labeling have revealed that the proepicardium contributes cells to the epicardium, subepicardial mesenchyme, coronaries, cardiac fibroblasts, and the mesenchyme of the atrioventricular valves (Gittenberger-de Groot et al. 1998; Manner, 1999; Perez-Pomares et al. 1997; Poelmann et al. 1993). Genetic Tbx18Cre and Wt1Cre lineage analyses confirmed the contribution of proepicardial cells to these populations (Cai et al. 2008; Zhou et al. 2008a). In the Tbx18Cre lineage analyses, reporter expression was visible in a subset of the vascular smooth muscle cells and cardiac fibroblasts, but not in the coronary endothelium. Unexpectedly, recombination also occurred in cardiomyocytes, suggesting that they, too, are derived from epicardium (Cai et al. 2008). However, the interpretation based on the Tbx18Cre lineage study was called into question by the demonstration that cardiomyocytes themselves express Tbx18 and that recombination in myocytes also occurred in mouse models in which epicardium formation was compromised (Christoffels et al. 2009). From the Wt1 Cre lineage analysis (Zhou et al. 2008a), it was observed that epicardium and subepicardial mesenchyme were positive for the reporter, as well as most of the smooth muscle cells and a minority of the endothelial cells of the coronaries. This analysis also showed patches of βGal-expressing cardiomyocytes located throughout the myocardium, which comprised ≈10% of the total amount of cardiomyocytes. This finding was unexpected because classic labeling analysis with the use of chicken–quail chimeras had never shown an epicardial contribution to the myocardium (Manner 1999) and Wt1 expression has not been reported in cardiomyocytes. The epicardial origin of progenitors contributing to the myocardium was further substantiated with the use of a tamoxifen inducible Cre
TCM Vol. 20, No. 1, 2010
Figure 2. Differentiation of precardiac mesoderm into proepicardial and myocardial cells. Precardiac mesoderm is induced to express Wt1 and the transcription factor Tbx18 by (unknown) signals secreted from the hepatic endoderm. Wt1- and Tbx18-expressing precardiac mesoderm separates into myocardial and proepicardial cells by a cooperative action of Bmp and Fgf signaling. Within the proepicardium and epicardium, mesenchymal cells are formed from epithelial cells by the process of EMT. The myocardium located cranially to the proepicardium expresses Tbx18. Via the proepicardium and subsequently the epicardium cells of the coronary vessels, cardiac fibroblast, epicardial-derived cells, and a small number of cardiomyocytes are added to the heart.
(CreERT2). This Cre enzyme becomes biologically active in the presence of tamoxifen. Upon activation of the Cre enzyme after formation of the embryon-
ic epicardium at E10.5 and E11.5, a very small number of reporter-positive cells could be found, compared with the analysis with the use of the Wt1Cre line.
Nevertheless, the reporter-positive cells were found in smooth muscle, endothelial, and myocardial cells, pointing to an epicardial origin of these cells. Alternatively, because recombination by Cre is considered very efficient, it may point to a low-level Wt1 expression, which is not reported, as yet, but sufficient to induce Cre-mediated recombination. The observed limited contribution of epicardially derived cells to the nonmyocardial component in the formed heart, as assessed in genetic lineage analyses compared with classic labeling analyses, is puzzling. In classic lineage analyses, all cells contributing to the coronaries, including endothelium, smooth muscle cells, and pericytes, as well as the majority, if not all, of intermyocardial fibroblasts, and a small population of the mesenchyme in the atrioventricular valve leaflets were found to be of proepicardial origin. The reason for the discrepancies between the classic and genetic lineage analyses might be related to (1) the invasive nature of quail proepicardial cells used in the classic analysis; (2) the onset of the genetic lineage tracing, showing the developmental history from the start of gestation onward; and (3) the shorter developmental window in which transplanted quail cells can be traced. Further studies will be required
Figure 3. Model of addition of progenitor cells to the developing heart. A, Undifferentiated cardiac progenitors (blue) proliferate outside the heart, in the pericardial back wall, and are formed by EMT. Progenitors are added to the inflow and outflow poles of the heart (black arrows) and differentiate into cardiomyocytes when passing a zone of high levels of Bmp2 (red). The proepicardium (green) develops just caudally of the zone of the highest level of Bmp2. B, In situ hybridization showing the pattern of expression of Bmp2, cranial to the proepicardium of a chicken embryo stage 16. C, Bypassing the zone of the highest level of Bmp2, via the formation of the proepicardium, enables the addition of cardiac progenitors (blue) to the developing heart.
TCM Vol. 20, No. 1, 2010
5
to properly analyze the epicardial-derived contribution to the (non)myocardial component of the heart. A further question that needs addressing is whether all cells of the proepicardium are able to contribute to the different lineages at later stages or if, within the proepicardium, a subset of cells is already committed to a specific-lineage.
The Epicardium as a Progenitor Population
The capacity of epicardial cells to contribute to a variety of cells in the adult heart, including myocardial cells, identifies the epicardial cells as a potentially interesting cell type in the search for progenitor cells. Epicardium-derived progenitor cells, the mesenchymal cells derived from the epicardium by EMT, are suggested to function as cardiac stem cells (Wessels and Perez-Pomares 2004; Winter et al. 2007). Another indication for the presence of epicardially derived cardiac progenitor cells is the capacity of mesoangioblasts present in the adult heart to enter the cardiac lineage (Galvez et al. 2008). Recently, it has been shown that during development Wt1 is required for cardiovascular progenitor cell formation through transcriptional control of Snail and E-cadherin in the epicardium (Martinez-Estrada et al. 2010). The presence of progenitor cells in the heart, originating from the epicardium, might indicate that these progenitors are added to the heart via the proepicardium during development. During early development, progenitors are added to the heart at both the inflow and outflow poles of the heart. These undifferentiated cardiac progenitors proliferate outside the heart (Van Den Berg et al. 2009) and are formed by the process of EMT (Martinez-Estrada et al. 2010). Epithelial-to-mesenchymal transformation has already been observed in the proepicardium (Carmona et al. 2001). Undifferentiated cardiac progenitors differentiate into myocardial cells when added, via the inflow or outflow pole, to the heart. Bmp signaling plays an important role in the addition of the undifferentiated cardiac progenitor to both poles of the heart (Prall et al. 2007). Cardiac progenitor cells proliferate in the presence of low levels of Bmp2 but differentiate into myocardial cells when passing a zone of higher concen-
6
trations of Bmp2 present at both the inflow and outflow poles of the heart (Prall et al. 2007; Van Wijk et al. 2007). The proepicardium develops just caudally of the zone of the highest levels of Bmp2 located at the inflow pole of the heart (Kruithof et al. 2006; Van Wijk et al. 2009). A subset of these progenitors could be added to the heart via the proepicardium, bypassing this zone and thus retaining their undifferentiated precardiac precursor state (Figure 3). In line with this, it has been shown in vitro that, in the presence of Bmp2 and Fgf2, the progenitors of proepicardia remain in an undifferentiated state (Kruithof et al. 2006; Van Wijk et al. 2009). The cooperative action of Bmp and Fgf may serve to keep these progenitors, also at later stages, in an undifferentiated and proliferative state. This idea is underscored by the presence of cKit-positive progenitors (Limana et al. 2007) and activated Bmp and Fgf signaling pathways in the subepicardial mesenchyme (Van Wijk et al. 2009).
Future Questions
The possibility that cardiac progenitor cells are added to the heart via the proepicardium raises several intriguing questions that need to be addressed: (1) Is the interaction of Bmp and Fgf signaling, which regulates the lineage separation of myocardial and epicardial cells during the development of the proepicardium, still operational in epicardially derived progenitors? (2) Do adult epicardial cells retain the capacity to form progenitor cells that can differentiate into myocardial cells? (3) If progenitors are added via the proepicardium, where are these progenitors “stored” in the formed heart? The common origin of myocardial and epicardial cells, and the signals that regulate the differentiation of progenitors into myocardial and nonmyocardial cells or that regulate the maintenance of a progenitor state in proepicardial-derived cells provide challenging inroads in the analysis of the existence, differentiation, and use of these potential progenitor cells.
Acknowledgments
The authors thank Prof Dr AFM Moorman, Dr VM Christoffels, and Dr P
Barnett for critically reading the manuscript. The work in the laboratory of Dr MJB van den Hoff is supported by Netherlands Heart Foundation grant 1996M002 and the European Community's Sixth Framework Program contract (“HeartRepair”) grant LSHM-CT2005-018630. References Buckingham M, Meilhac S & Zaffran S: 2005. Building the mammalian heart from two sources of myocardial cells. Nat Rev Genet 6:826–837. Cai CL, Liang X, Shi Y, et al: 2003. Isl1 identifies a cardiac progenitor population that proliferates prior to differentiation and contributes a majority of cells to the heart. Dev Cell 5:877–889. Cai CL, Martin JC, Sun Y, et al: 2008. A myocardial lineage derives from Tbx18 epicardial cells. Nature 454:104–108. Carmona R, Gonzalez-Iriarte M, PerezPomares JM, et al: 2001. Localization of the Wilm's tumour protein Wt1 in avian embryos. Cell Tissue Res 303:173–186. Christoffels VM, Grieskamp T, Norden J, et al: 2009. Tbx18 and the fate of epicardial progenitors. Nature 458:E8–E9. Christoffels VM, Mommersteeg MTM, Trowe MO, et al: 2006. Formation of the venous pole of the heart from an Nkx2-5-negative precursor population requires Tbx18. Circ Res 98:1555–1563. Dettman RW, Denetclaw W, Ordahl CP, et al: 1998. Common epicardial origin of coronary vascular smooth muscle, perivascular fibroblasts, and intermyocardial fibroblasts in the avian heart. Dev Biol 193:169–181. Di Meglio F, Castaldo C, Nurzynska D, et al: 2009. Epicardial cells are missing from the surface of hearts with ischemic cardiomyopathy: a useful clue about the self-renewal potential of the adult human heart? Int J Cardiol; 2009 Jan 26 [Epub ahead of print]. Galvez BG, Sampaolesi M, Barbuti A, et al: 2008. Cardiac mesoangioblasts are committed, self-renewable progenitors, associated with small vessels of juvenile mouse ventricle. Cell Death Differ 15:1417–1428. Gittenberger-de Groot AC, Vrancken Peeters MPFM, Mentink MMT, et al: 1998. Epicardium-derived cells contribute a novel population to the myocardial wall and the atrioventricular cushions. Circ Res 82: 1043–1052. Haenig B & Kispert A: 2004. Analysis of TBX18 expression in chick embryos. Dev Genes Evol 214:407–411. Hirose T, Karasawa M, Sugitani Y, et al: 2006. PAR3 is essential for cyst-mediated epicardial development by establishing apical cortical domains. Development 133: 1389–1398.
TCM Vol. 20, No. 1, 2010
Ishii Y, Langberg JD, Hurtado R, et al: 2007. Induction of proepicardial marker gene expression by the liver bud. Development 134:3627–3637.
tinuous formation of coronary vessels. Proc Natl Acad Sci U S A 89:9504–9508.
Kraus F, Haenig B & Kispert A: 2001. Cloning and expression analysis of the mouse T-box gene Tbx18. Mech Dev 100:83–86.
Moore AW, McInnes L, Kreidberg J, et al: 1999. YAC complementation shows a requirement for Wt1 in the development of epicardium, adrenal gland and throughout nephrogenesis. Development 126:1845–1857.
Kruithof BPT, van Wijk B, Somi S, et al: 2006. BMP and FGF regulate the differentiation of multipotential pericardial mesoderm into the myocardial or epicardial lineage. Dev Biol 295:507–522.
Perez-Pomares JM, Macias D, Garcia-Garrido L, et al: 1997. Contribution of the primitive epicardium to the subepicardial mesenchyme in hamster and chick embryos. Dev Dyn 210:96–105.
Lepilina A, Coon AN, Kikuchi K, et al: 2006. A dynamic epicardial injury response supports progenitor cell activity during zebrafish heart regeneration. Cell 127: 607–619.
Poelmann RE, Gittenberger-de Groot AC, Mentink MMT, et al: 1993. Development of the cardiac coronary vascular endothelium, studied with anti-endothelial antibodies in chicken-quail chimeras. Circ Res 73: 559–568.
Limana F, Bertolami C, Mangoni A, et al: 2009. Myocardial infarction induces embryonic reprogramming of epicardial c-kit (+) cells: role of the pericardial fluid. JMCC 48:609–618. Limana F, Zacheo A, Mocini D, et al: 2007. Identification of myocardial and vascular precursor cells in human and mouse epicardium. Circ Res 101:1255–1265. Ma Q, Zhou B & Pu WT: 2008. Reassessment of Isl1 and Nkx2-5 cardiac fate maps using a Gata4-based reporter of Cre activity. Dev Biol 323:98–104. Manner J: 1999. Does the subepicardial mesenchyme contribute myocardioblasts to the myocardium of the chick embryo heart? A quail–chick chimera study tracing the fate of the epicardial primordium. Anat Rec 255: 212–226. Manner J, Perez-Pomares JM, Macias D, et al: 2001. The origin, formation and developmental significance of the epicardium: a review. Cells Tissues Organs 169:89–103. Martinez-Estrada OM, Lettice LA, Essafi A, et al: 2010. Wt1 is required form cardiovascular progenitor cell formation through transcriptional control of Snail and Ecadherin. Nat Genet 42:89–93. Mikawa T & Fischman DA: 1992. Retroviral analysis of cardiac morphogenesis: discon-
TCM Vol. 20, No. 1, 2010
Prall OWJ, Menon MK, Solloway MJ, et al: 2007. An Nkx2-5/Bmp2/Smad1 negative feedback loop controls heart progenitor specification and proliferation. Cell 128: 947–959. Rochais F, Mesbah K & Kelly RG: 2009. Signaling pathways controlling second heart field development. Circ Res 104: 933–942.
progenitor cells conferred by a 3′UTR-iresCre allele of the homeobox gene Nkx2-5. Int J Dev Bank 46:431–439. Van Den Berg G, Abu-Issa R, De Boer BA, et al: 2009. A caudal proliferating growth center contributes to both poles of the forming heart tube. Circ Res 104:179–188. Van Wijk B, Moorman AFM & Van Den Hoff MJB: 2007. Role of bone morphogenetic proteins in cardiac differentiation. Cardiovasc Res 74:244–255. Van Wijk B, Van Den Berg G, Abu-Issa R, et al: 2009. Epicardium and myocardium separate from a common precursor pool by crosstalk between bone morphogenetic protein- and fibroblast growth factor-signaling pathways. Circ Res 105:431–441. Wessels A & Perez-Pomares JM: 2004. The epicardium and epicardially derived cells (EPDCs) as cardiac stem cells. Anat Rec 276A:43–57. Wiese C, Grieskamp T, Airik R, et al: 2009. Formation of the sinus node head and differentiation of sinus node myocardium are independently regulated by Tbx18 and Tbx3. Circ Res 104:388–397.
Schlueter J & Brand T: 2009. A right-sided pathway involving FGF8/Snail controls asymmetric development of the proepicardium in the chick embryo. Proc Natl Acad Sci U S A 106:7485–7490.
Winter EM, Grauss RW, Hogers B, et al: 2007. Preservation of left ventricular function and attenuation of remodeling after transplantation of human epicardium-derived cells into the infarcted mouse heart. Circulation 116:917–927.
Schlueter J, Manner J & Brand T: 2006. BMP is an important regulator of proepicardial identity in the chick embryo. Dev Biol 295: 546–558.
Zhou B, Ma Q, Rajagopal S, et al: 2008a. Epicardial progenitors contribute to the cardiomyocyte lineage in the developing heart. Nature 454:109–113.
Schulte I, Schlueter J, Abu-Issa R, et al: 2007. Morphological and molecular left-right asymmetries in the development of the proepicardium: a comparative analysis on mouse and chick embryos. Dev Dyn 236: 684–695.
Zhou B, Von Gise A, Ma Q, et al: 2008b. Nkx25- and Isl1-expressing cardiac progenitors contribute to proepicardium. Biochem Biophys Res Commun 375:450–453.
Soriano P: 1999. Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet 21:70–71. Stanley EG, Biben C, Elefanty A, et al: 2002. Efficient Cre-mediated deletion in cardiac
PII S1050-1738(10)00029-0
TCM
7
During development, the epicardium, an epithelial layer that covers the heart, gives rise to a large portion of the nonmyocardial cells present in the heart. The epicardium arises from a structure, called the proepicardium, which forms at the inflow of the developing heart. By epithelial-tomesenchymal transformation, mesenchymal cells are formed that will subsequently populate the stroma of the proepicardium and the subepicardium. Based on labeling analysis, the proepicardium and part of the myocardium have been shown to be derived from a common cardiogenic precursor population. In this review, we will discuss the common cardiogenic origin of proepicardial and myocardial cells, the underlying processes and factors that play a role in the separation of the lineages, and their potential role in cardiac regenerative approaches. (Trends Cardiovasc Med 2010;20:1–7) n 2010, Elsevier Inc.
Introduction
Given the limited regenerative capacity of the adult heart, cardiac damage frequently progresses to congestive heart failure. Attempts to stimulate cardiac repair have seen large efforts being made to identify and isolate cardiac progenitors. Several lines of evidence suggest that the epicardium, the nonmyocardial epithelium covering the myocardium, could conceivably play a role in cardiac regeneration. In zebrafish, reac-
Bram van Wijk and Maurice van den Hoff are at the Heart Failure Research Center, Academic Medical Center, 1105AZ Amsterdam, The Netherlands. ⁎ Address correspondence to: M.J.B. van den Hoff, PhD, Academic Medical Center, Department of Anatomy, Embryology & Physiology, Meibergdreef 15, 1105AZ Amsterdam, The Netherlands. Tel.: (+31) 205665415; fax: (+31) 206976177; e-mail: m.j.vandenhoff@ amc.uva.nl. © 2010, Elsevier Inc. All rights reserved. 1050-1738/$-see front matter
TCM Vol. 20, No. 1, 2010
tivation of the epicardium plays a critical role in the regeneration of injured myocardium (Lepilina et al. 2006). Although the mammalian heart possesses little regenerative capacity in contrast to the heart of zebrafish, its epicardium has also been suggested to be involved in myocardial regeneration (Di Meglio et al. 2009; Limana et al. 2007, 2009; Winter et al. 2007). During development, the epicardium arises from a cauliflower-like structure called the proepicardium. Around the third day of development (Hamburger– Hamilton stage 16) in chicken, 9.5 days of development in mouse, and ≈3.5 weeks (Carnegie stage 10) of human development, the proepicardium arises from splanchnic mesoderm that covers the vitelline veins at the inflow of the developing heart (Manner et al. 2001). Until this stage, the heart lies “naked” within the pericardial cavity. The proepicardium protrudes into the pericardial cavity and contacts the myocardium of the atrioventricular canal. Subsequently, epicardial cells migrate over the heart
tube to cover the myocardium. Within the proepicardium and later also in the epicardium that covers the heart tube, a subset of the epithelial cells undergoes epithelial-to-mesenchymal transformation (EMT) (Wessels and Perez-Pomares 2004). These mesenchymal cells provide a large portion of the nonmyocardial cells of the heart, for instance, cardiac fibroblasts, coronary smooth muscle cells, and coronary endothelial cells. Although still an issue of debate (Christoffels et al. 2009), recent genetic labeling studies suggest that cells derived from the epicardium might contribute to the myocardial lineage, as well (Cai et al. 2008; Zhou et al. 2008a). The wide variety of cells the proepicardium contributes to the heart (Cai et al. 2008; Gittenberger-de Groot et al. 1998; Manner 1999; Perez-Pomares et al. 1997; Wessels and Perez-Pomares, 2004; Zhou et al. 2008a), coupled with the fact that the (pro)epicardium and myocardium share a common cardiogenic precursor (Van Wijk et al. 2009; Zhou et al. 2008a,b), makes epicardial cells a potential source of cardiac progenitor cells and as such may serve a potential source of cells for regenerative approaches. In this review, we will discuss the common origin of epicardial and myocardial cells, the processes and factors that play a role in the separation of myocardial and nonmyocardial lineages, and the potential role of epicardial-derived cells as progenitor cells.
Common Origin of Epicardial and Myocardial Cells
Classic Lineage Tracing Analyses The heart is formed from cells of the splanchnic mesoderm. The bilateral zones of the splanchnic mesoderm, which will derive the heart, are referred to as the heart-forming regions. Within these regions, cells have been subdivided into two developmental fields: the first and second heart fields (Buckingham et al. 2005). This distinction should be considered a spatiotemporal separation
1
of cells within a greater heart-forming region (Rochais et al. 2009). The lateral cells of this region, the first heart field, are the first to exit the cell cycle, subsequently differentiating into myocardium and giving rise to the primary heart tube. The cells located medially and caudally within the heart-forming region are referred to as the second heart field (Figure 1). These cells continue to proliferate in an undifferentiated state after the formation of the linear heart tube and are gradually added to both the inflow and outflow regions of the heart (Van Den Berg et al. 2009) as it loops. At the inflow of the heart, these progenitors contribute both myocardial and nonmyocardial cells. The nonmyocardial component of the heart is largely derived from cells of the proepicardium that develops at the inflow of the heart. In the cardiac progenitor pool, present upstream of the heart tube before the induction of the proepicardium, a lineage decision takes place, defining cells that will differentiate into cardiomyocytes and be added to the venous pole of the heart or cells that will differentiate into the proepicardial lineage (Van Wijk et al. 2009). An in-depth study of the various cell types contributing to the inflow of the heart and the proepicardium has long been lacking. Classic lineage analyses, with the use of vital dyes, radioactive deposits, or retroviruses, were hampered because the venous pole of the heart is difficult to approach in the living embryo owing to technical limitations. Only recently have we been able to demonstrate the contribution of the second heart field to both the inflow myocardium and the proepicardium with the use of a vital dye labeling (Van Wijk et al. 2009). In this analysis, a small group of splanchnic mesodermal cells, expressing the T-box transcription factor 18 (Tbx18) at the inflow region of the heart, was labeled 1 day before the initiation of proepicardium formation (stage 11). At the time of labeling, these cells neither were positive for myocardial markers, like cardiac troponin I or myosin heavy chain isoforms, nor showed morphologic characteristics of proepicardium formation. Twenty-four hours later, the label was detected both in myocardial cells of the inflow tract and in proepicardial cells. Because, in this experiment, a small group of cells was labeled rather
2
than a single cell, it cannot be excluded that the labeled population comprises a mixed population instead of bipotential progenitor cells. However, this would seem unlikely, as gene expression analysis of the mesoderm at the stage of labeling, that is, before formation of the proepicardium, displays no heterogeneity in gene expression. Earlier cell tracing studies in which (1) quail proepicardia were transplanted into chicken embryos (Manner 1999; Perez-Pomares et al. 1997), (2) adenovirus-labeled chicken proepicardia were transplanted into chicken acceptors (Dettman et al. 1998), and (3) proepicardia were retrovirus-labeled in ovo (Mikawa and Fischman, 1992) were unable to demonstrate proepicardial contribution to the myocardium. Although cell tracing analysis revealed no contribution of proepicardial cells to the myocardium, proepicardial cells have been shown to differentiate into heart muscle cells in vitro (Kruithof et al. 2006; Schlueter et al. 2006; Van Wijk et al. 2009). The two most important differences between the recent labeling experiments and the earlier analyses are the developmental stage at which the cells were labeled and the techniques used to label the proepicardia. In the recent experiment, the mesoderm upstream of the forming heart tube was labeled before the induction and formation of the proepicardium at stage 11, whereas in the earlier experiments, the proepicardium was labeled later, at stage 16 to 17. Second, in the studies in which proepicardia were transplanted, the donor tissue was not in direct contact with the inflow of the recipient embryo. Consequently, the physical barrier, that is, the pericardial cavity, prevented the transplanted cells from contributing to the inflow myocardium. The expression of Tbx18 first becomes evident in the splanchnic layer immediately upstream of the heart tube at stage 11 (Haenig and Kispert 2004; Van Wijk et al. 2009). One day later during development, Tbx18 expression can be observed in distinct regions at the inflow of the heart, being (1) myocardial cells just cranial to the base of the proepicardium (Kruithof et al. 2006; Van Wijk et al. 2009); (2) sinus horn myocardium, that is, the myocardial sleeves covering the cardinal veins (Christoffels et al. 2006); and (3) proepicardial cells (Haenig and
Kispert 2004; Kraus et al. 2001). Histologic analysis of the proepicardium has shown that the proepicardial villi are sheathed in epithelial cells and filled with mesenchymal cells and extracellular matrix. Because of the presence of genes involved in EMT, like Wt1 and Snail, in the proepicardium (Carmona et al. 2001), it is likely that during proepicardium formation epithelial cells transform into mesenchymal cells that invade the proepicardial matrix. Within the proepicardium, several genes have been reported to be heterogeneously expressed, for example, P-Erk (Van Wijk et al. 2009), FGF2 (Kruithof et al. 2006), Flk1 (Cai et al. 2008), PAR3, and ZO1 (Hirose et al. 2006), indicating that the proepicardium is a heterogeneous population of cells. Taken together, a homogeneous, Tbx18-expressing mesodermal cell population upstream of the inflow of the heart, which covers the vitelline veins at stage 11, gives rise to proepicardial and inflow myocardial cells. Within the proepicardium, epithelial cells transform into mesenchymal cells that will subsequently invade the proepicardial matrix. Genetic Lineage Analyses The development of a two-component mouse system, in which one mouse harbors a conditional reporter gene, like lacZ or GFP, and the other mouse expresses Cre (Causes recombination), has enabled genetic lineage tracing analyses to be carried out (Soriano 1999). The last couple of years have provided a variety of different Cre lines that have been used to analyze the development of the inflow of the heart. The Nkx2.5IRES-Cre, in which an IRESCre cassette is inserted into the 3′UTR of the Nkx2.5 gene (Stanley et al. 2002), showed robust reporter gene expression in all myocardial cells and the proepicardium, but not in sinus horn myocardium (Christoffels et al. 2006; Stanley et al. 2002). Similar results were obtained with the use of an Nkx2.5Cre line (Zhou et al. 2008b) or an Isl1Cre line (Cai et al. 2003; Ma et al. 2008), in which Cre is knocked in at the translation start sites. In situ hybridization and immunohistochemical analyses of the inflow tract have shown that neither the proepicardium nor the sinus horn myocardium expresses Nkx2.5 or Isl1 (Christoffels et
TCM Vol. 20, No. 1, 2010
Figure 1. Initial heart formation. Panels A, B, and C spatially illustrate early heart formation in chicken from the ventral side. A, The heartforming regions (HFR) are indicated in gray (primary heart field, Nkx2.5 lineage) and blue (second heart field, Isl1 lineage). The dotted red line indicates the area of the HFR that will express Wt1 and the transcription factor Tbx18. B, The bilateral HFR adjoin in the midline by folding of the embryonic disc (hatched arrows). The red line indicates the area of the HFR that expresses Wt1 and Tbx18. C, Progressive folding lengthens the heart tube in caudal direction, forms the foregut, and establishes the pericardial back wall.
al. 2006; Ma et al. 2008; Zhou et al. 2008b). These findings reveal that cells of the proepicardium and sinus horn myocardium are derived from progenitors that have at some point expressed Nkx2.5 and Isl1 during development, though not necessarily at the same time. This suggest that both proepicardium and sinus horn progenitors are derived from the second heart field, but turn off isl1 earlier, and do not activate nkx2.5 like other second heart field progenitors. However, the relation between the proepicardial and sinus horn progenitors is currently unclear. Indeed, sinus horn myocardium is formed subsequent to the formation of the proepicardium, that is, from E9.5 onward, and, in contrast to epicardium and all other myocardium, is unrecombined in Nkx2.5IRES-Cre;R26R embryos, suggesting a much briefer period of Nkx2.5 expression in these progenitors compared with the proepicardial progenitors
TCM Vol. 20, No. 1, 2010
(Christoffels et al. 2006). Consequently, the myocardium located directly cranial to the proepicardium is different from sinus horn myocardium. In the neonatal heart, the myocardium directly cranial to the proepicardium finds its destiny in the myocardium of the sinus node and right venous valve (Wiese et al. 2009). Because the insertion of Cre in the Nkx2.5Cre and Isl1Cre lines disrupts expression of functional Nkx2.5 and Isl1 protein, respectively, their role in proepicardial development could be tested. Homozygous disruption of Nkx2.5 or Isl1 results in growth retardation and cardiac malformation. In Nkx2.5Cre/Cre mice, the proepicardium fails to develop, whereas in Isl1Cre/Cre, the proepicardium remains underdeveloped (Zhou et al. 2008b). These findings suggest that Nkx2.5, although not expressed in the proepicardium, is required for its formation, this not being the case for Isl1. Isl1 seems to be required for proper expan-
sion of the cardiac progenitor pool (Prall et al. 2007) and consequently for providing sufficient progenitors for proepicardial development. Analysis of Tbx18Cre mice revealed recombination in the forming epicardium and adult heart (Cai et al. 2008). Wilms tumor 1 (Wt1)-Cre, another Cre line of importance in (pro)epicardial development, demonstrated a similar pattern of recombination, that is, in the epicardium and in cells of the adult heart (Zhou et al. 2008a) (details of both Cre lines will be discussed later in this review). The contribution of the Tbx18Cre and Wt1Cre lineage to the inflow myocardium has not been addressed in these studies. Wt1-expressing proepicardial cells were shown to be derived from cells that were found to belong to both the Nkx2-5 and Isl1 lineages, even though Wt1 protein was never found to be coexpressed with Isl1 nor Nkx2-5 proteins (Zhou et al. 2008a).
3
Taken together, labeling studies of Tbx18-expressing progenitors (Van Wijk et al. 2009), together with lineage analyses with the use of Isl1- and Nkx2-5-Cre lines (Ma et al. 2008; Zhou et al. 2008b), have identified a common origin of myocardial and (pro)epicardial cells.
Regulation of the Separation of Myocardial and Nonmyocardial Cells
The earliest markers that identify the myocardial and proepicardial progenitor population are Tbx18 and Wt1. Expression of Tbx18 and Wt1 starts just before E8.0 in mouse (Kraus et al. 2001; Zhou et al. 2008a) and stage 11 in chicken (Haenig and Kispert, 2004; Van Wijk et al. 2009). Both in mouse (E9.5) and chicken (stage 16), the proepicardium starts to become morphologically evident as villous structures protruding into the pericardial cavity (Schulte et al. 2007). Proepicardial villi appear on both the left and right vitelline veins. At the right side, the villi expand and form the proepicardium, whereas at the left side, in chicken, the villi undergo apoptosis and disappear. The pathways regulating this left–right asymmetry are beyond the scope of this review and have been discussed elsewhere (Schlueter and Brand, 2009). By contrast, in mouse, the villi expand and form a proepicardium that is located medially (Schulte et al. 2007). The factors that induce expression of Tbx18 and Wt1 are presently unknown. However, xenotransplantion of stage 17 quail liver buds to a caudal location in chicken of various developmental stages showed induction of Tbx18 and/or Wt1 expression in the adjacent mesoderm from stage 12 onward, indicating that secreted factors derived from the hepatic endoderm induce the expression of Tbx18 and Wt1. Similarly transplanting lung buds, as a control, did not have this effect, hinting that the forming hepatic endoderm is the probable source of factors that induce expression of Tbx18 and Wt1 in the splanchnic mesoderm. Because lung buds produce Bmp and Fgf, these growth factors are unlikely candidates to induce the expression of Tbx18 and Wt1 (Ishii et al. 2007; Van Wijk et al. 2009). Although Tbx18 marks the progenitor population of proepicardial and inflow myocardial cells, its expression is not essential for the formation of the proepicardium because func-
4
tional disruption of Tbx18 does not result in aberrant proepicardial development (Christoffels et al. 2006). Homozygous deletion of Wt1, on the other hand, severely impairs (pro)epicardial development (Moore et al. 1999). Wt1's involvement in EMT suggests that EMT plays an important role in the formation of proepicardial cells (Carmona et al. 2001). Besides EMT, the regulation of cell polarity also seems to be involved in proepicardium formation, as deletion of the mammalian homologue of the Caenorhabditis elegans polarity protein, Par3, results in defective (pro)epicardial development (Hirose et al. 2006). The factors that regulate the division of the progenitor population into myocardium on the one hand and proepicardium on the other are largely unknown. Although Bmp and Fgf do not induce the expression of Tbx18 or Wt1 in this progenitor population (Ishii et al. 2007), they do play a role in the separation of myocardial and nonmyocardial cells from this population (Van Wijk et al. 2009). Exposing embryos to Bmp2, Fgf2, and/or the Mek1 inhibitor U0126 has shown that Bmp2 and Fgf2 signaling pathways cooperate in the separation and differentiation of the Tbx18-positive progenitor pool into cardiomyocytes and proepicardial cells. Smad-mediated Bmp-signaling directs the progenitors into the myocardial lineage. Erk-mediated Fgf2 signaling, on the one hand, directs the progenitors into the proepicardial lineage and blunts the differentiation of the progenitors into the myocardial lineage (Van Wijk et al. 2009). The inhibition of myocardial differentiation of the progenitors by Fgf2 was found to be the result of disrupting Smad-mediated Bmp-signaling by phosphorylating receptor Smad, such that it cannot interact with common Smad4 and thus accumulates in the cytoplasm. As a consequence, the initiated Bmp signal is not conveyed to the nucleus, thereby disrupting the Bmp response (Figure 2) (Van Wijk et al. 2009). Taken together, these data indicate that (an) unknown signal(s) secreted from the hepatic endoderm induces Tbx18 and Wt1 expression in the precardiac progenitor population. Separation of this progenitor population into the myocardial and epicardial lineage is regulated by the cooperative interaction
between Bmp and Fgf-signaling (Ishii et al. 2007; Schlueter et al. 2006; Van Wijk et al. 2009).
Proepicardial Contribution to the Heart
Xenotransplantation studies with the use of the quail–chick chimera technique as well as cell fate analyses with the use of retroviral and adenoviral cell labeling have revealed that the proepicardium contributes cells to the epicardium, subepicardial mesenchyme, coronaries, cardiac fibroblasts, and the mesenchyme of the atrioventricular valves (Gittenberger-de Groot et al. 1998; Manner, 1999; Perez-Pomares et al. 1997; Poelmann et al. 1993). Genetic Tbx18Cre and Wt1Cre lineage analyses confirmed the contribution of proepicardial cells to these populations (Cai et al. 2008; Zhou et al. 2008a). In the Tbx18Cre lineage analyses, reporter expression was visible in a subset of the vascular smooth muscle cells and cardiac fibroblasts, but not in the coronary endothelium. Unexpectedly, recombination also occurred in cardiomyocytes, suggesting that they, too, are derived from epicardium (Cai et al. 2008). However, the interpretation based on the Tbx18Cre lineage study was called into question by the demonstration that cardiomyocytes themselves express Tbx18 and that recombination in myocytes also occurred in mouse models in which epicardium formation was compromised (Christoffels et al. 2009). From the Wt1 Cre lineage analysis (Zhou et al. 2008a), it was observed that epicardium and subepicardial mesenchyme were positive for the reporter, as well as most of the smooth muscle cells and a minority of the endothelial cells of the coronaries. This analysis also showed patches of βGal-expressing cardiomyocytes located throughout the myocardium, which comprised ≈10% of the total amount of cardiomyocytes. This finding was unexpected because classic labeling analysis with the use of chicken–quail chimeras had never shown an epicardial contribution to the myocardium (Manner 1999) and Wt1 expression has not been reported in cardiomyocytes. The epicardial origin of progenitors contributing to the myocardium was further substantiated with the use of a tamoxifen inducible Cre
TCM Vol. 20, No. 1, 2010
Figure 2. Differentiation of precardiac mesoderm into proepicardial and myocardial cells. Precardiac mesoderm is induced to express Wt1 and the transcription factor Tbx18 by (unknown) signals secreted from the hepatic endoderm. Wt1- and Tbx18-expressing precardiac mesoderm separates into myocardial and proepicardial cells by a cooperative action of Bmp and Fgf signaling. Within the proepicardium and epicardium, mesenchymal cells are formed from epithelial cells by the process of EMT. The myocardium located cranially to the proepicardium expresses Tbx18. Via the proepicardium and subsequently the epicardium cells of the coronary vessels, cardiac fibroblast, epicardial-derived cells, and a small number of cardiomyocytes are added to the heart.
(CreERT2). This Cre enzyme becomes biologically active in the presence of tamoxifen. Upon activation of the Cre enzyme after formation of the embryon-
ic epicardium at E10.5 and E11.5, a very small number of reporter-positive cells could be found, compared with the analysis with the use of the Wt1Cre line.
Nevertheless, the reporter-positive cells were found in smooth muscle, endothelial, and myocardial cells, pointing to an epicardial origin of these cells. Alternatively, because recombination by Cre is considered very efficient, it may point to a low-level Wt1 expression, which is not reported, as yet, but sufficient to induce Cre-mediated recombination. The observed limited contribution of epicardially derived cells to the nonmyocardial component in the formed heart, as assessed in genetic lineage analyses compared with classic labeling analyses, is puzzling. In classic lineage analyses, all cells contributing to the coronaries, including endothelium, smooth muscle cells, and pericytes, as well as the majority, if not all, of intermyocardial fibroblasts, and a small population of the mesenchyme in the atrioventricular valve leaflets were found to be of proepicardial origin. The reason for the discrepancies between the classic and genetic lineage analyses might be related to (1) the invasive nature of quail proepicardial cells used in the classic analysis; (2) the onset of the genetic lineage tracing, showing the developmental history from the start of gestation onward; and (3) the shorter developmental window in which transplanted quail cells can be traced. Further studies will be required
Figure 3. Model of addition of progenitor cells to the developing heart. A, Undifferentiated cardiac progenitors (blue) proliferate outside the heart, in the pericardial back wall, and are formed by EMT. Progenitors are added to the inflow and outflow poles of the heart (black arrows) and differentiate into cardiomyocytes when passing a zone of high levels of Bmp2 (red). The proepicardium (green) develops just caudally of the zone of the highest level of Bmp2. B, In situ hybridization showing the pattern of expression of Bmp2, cranial to the proepicardium of a chicken embryo stage 16. C, Bypassing the zone of the highest level of Bmp2, via the formation of the proepicardium, enables the addition of cardiac progenitors (blue) to the developing heart.
TCM Vol. 20, No. 1, 2010
5
to properly analyze the epicardial-derived contribution to the (non)myocardial component of the heart. A further question that needs addressing is whether all cells of the proepicardium are able to contribute to the different lineages at later stages or if, within the proepicardium, a subset of cells is already committed to a specific-lineage.
The Epicardium as a Progenitor Population
The capacity of epicardial cells to contribute to a variety of cells in the adult heart, including myocardial cells, identifies the epicardial cells as a potentially interesting cell type in the search for progenitor cells. Epicardium-derived progenitor cells, the mesenchymal cells derived from the epicardium by EMT, are suggested to function as cardiac stem cells (Wessels and Perez-Pomares 2004; Winter et al. 2007). Another indication for the presence of epicardially derived cardiac progenitor cells is the capacity of mesoangioblasts present in the adult heart to enter the cardiac lineage (Galvez et al. 2008). Recently, it has been shown that during development Wt1 is required for cardiovascular progenitor cell formation through transcriptional control of Snail and E-cadherin in the epicardium (Martinez-Estrada et al. 2010). The presence of progenitor cells in the heart, originating from the epicardium, might indicate that these progenitors are added to the heart via the proepicardium during development. During early development, progenitors are added to the heart at both the inflow and outflow poles of the heart. These undifferentiated cardiac progenitors proliferate outside the heart (Van Den Berg et al. 2009) and are formed by the process of EMT (Martinez-Estrada et al. 2010). Epithelial-to-mesenchymal transformation has already been observed in the proepicardium (Carmona et al. 2001). Undifferentiated cardiac progenitors differentiate into myocardial cells when added, via the inflow or outflow pole, to the heart. Bmp signaling plays an important role in the addition of the undifferentiated cardiac progenitor to both poles of the heart (Prall et al. 2007). Cardiac progenitor cells proliferate in the presence of low levels of Bmp2 but differentiate into myocardial cells when passing a zone of higher concen-
6
trations of Bmp2 present at both the inflow and outflow poles of the heart (Prall et al. 2007; Van Wijk et al. 2007). The proepicardium develops just caudally of the zone of the highest levels of Bmp2 located at the inflow pole of the heart (Kruithof et al. 2006; Van Wijk et al. 2009). A subset of these progenitors could be added to the heart via the proepicardium, bypassing this zone and thus retaining their undifferentiated precardiac precursor state (Figure 3). In line with this, it has been shown in vitro that, in the presence of Bmp2 and Fgf2, the progenitors of proepicardia remain in an undifferentiated state (Kruithof et al. 2006; Van Wijk et al. 2009). The cooperative action of Bmp and Fgf may serve to keep these progenitors, also at later stages, in an undifferentiated and proliferative state. This idea is underscored by the presence of cKit-positive progenitors (Limana et al. 2007) and activated Bmp and Fgf signaling pathways in the subepicardial mesenchyme (Van Wijk et al. 2009).
Future Questions
The possibility that cardiac progenitor cells are added to the heart via the proepicardium raises several intriguing questions that need to be addressed: (1) Is the interaction of Bmp and Fgf signaling, which regulates the lineage separation of myocardial and epicardial cells during the development of the proepicardium, still operational in epicardially derived progenitors? (2) Do adult epicardial cells retain the capacity to form progenitor cells that can differentiate into myocardial cells? (3) If progenitors are added via the proepicardium, where are these progenitors “stored” in the formed heart? The common origin of myocardial and epicardial cells, and the signals that regulate the differentiation of progenitors into myocardial and nonmyocardial cells or that regulate the maintenance of a progenitor state in proepicardial-derived cells provide challenging inroads in the analysis of the existence, differentiation, and use of these potential progenitor cells.
Acknowledgments
The authors thank Prof Dr AFM Moorman, Dr VM Christoffels, and Dr P
Barnett for critically reading the manuscript. The work in the laboratory of Dr MJB van den Hoff is supported by Netherlands Heart Foundation grant 1996M002 and the European Community's Sixth Framework Program contract (“HeartRepair”) grant LSHM-CT2005-018630. References Buckingham M, Meilhac S & Zaffran S: 2005. Building the mammalian heart from two sources of myocardial cells. Nat Rev Genet 6:826–837. Cai CL, Liang X, Shi Y, et al: 2003. Isl1 identifies a cardiac progenitor population that proliferates prior to differentiation and contributes a majority of cells to the heart. Dev Cell 5:877–889. Cai CL, Martin JC, Sun Y, et al: 2008. A myocardial lineage derives from Tbx18 epicardial cells. Nature 454:104–108. Carmona R, Gonzalez-Iriarte M, PerezPomares JM, et al: 2001. Localization of the Wilm's tumour protein Wt1 in avian embryos. Cell Tissue Res 303:173–186. Christoffels VM, Grieskamp T, Norden J, et al: 2009. Tbx18 and the fate of epicardial progenitors. Nature 458:E8–E9. Christoffels VM, Mommersteeg MTM, Trowe MO, et al: 2006. Formation of the venous pole of the heart from an Nkx2-5-negative precursor population requires Tbx18. Circ Res 98:1555–1563. Dettman RW, Denetclaw W, Ordahl CP, et al: 1998. Common epicardial origin of coronary vascular smooth muscle, perivascular fibroblasts, and intermyocardial fibroblasts in the avian heart. Dev Biol 193:169–181. Di Meglio F, Castaldo C, Nurzynska D, et al: 2009. Epicardial cells are missing from the surface of hearts with ischemic cardiomyopathy: a useful clue about the self-renewal potential of the adult human heart? Int J Cardiol; 2009 Jan 26 [Epub ahead of print]. Galvez BG, Sampaolesi M, Barbuti A, et al: 2008. Cardiac mesoangioblasts are committed, self-renewable progenitors, associated with small vessels of juvenile mouse ventricle. Cell Death Differ 15:1417–1428. Gittenberger-de Groot AC, Vrancken Peeters MPFM, Mentink MMT, et al: 1998. Epicardium-derived cells contribute a novel population to the myocardial wall and the atrioventricular cushions. Circ Res 82: 1043–1052. Haenig B & Kispert A: 2004. Analysis of TBX18 expression in chick embryos. Dev Genes Evol 214:407–411. Hirose T, Karasawa M, Sugitani Y, et al: 2006. PAR3 is essential for cyst-mediated epicardial development by establishing apical cortical domains. Development 133: 1389–1398.
TCM Vol. 20, No. 1, 2010
Ishii Y, Langberg JD, Hurtado R, et al: 2007. Induction of proepicardial marker gene expression by the liver bud. Development 134:3627–3637.
tinuous formation of coronary vessels. Proc Natl Acad Sci U S A 89:9504–9508.
Kraus F, Haenig B & Kispert A: 2001. Cloning and expression analysis of the mouse T-box gene Tbx18. Mech Dev 100:83–86.
Moore AW, McInnes L, Kreidberg J, et al: 1999. YAC complementation shows a requirement for Wt1 in the development of epicardium, adrenal gland and throughout nephrogenesis. Development 126:1845–1857.
Kruithof BPT, van Wijk B, Somi S, et al: 2006. BMP and FGF regulate the differentiation of multipotential pericardial mesoderm into the myocardial or epicardial lineage. Dev Biol 295:507–522.
Perez-Pomares JM, Macias D, Garcia-Garrido L, et al: 1997. Contribution of the primitive epicardium to the subepicardial mesenchyme in hamster and chick embryos. Dev Dyn 210:96–105.
Lepilina A, Coon AN, Kikuchi K, et al: 2006. A dynamic epicardial injury response supports progenitor cell activity during zebrafish heart regeneration. Cell 127: 607–619.
Poelmann RE, Gittenberger-de Groot AC, Mentink MMT, et al: 1993. Development of the cardiac coronary vascular endothelium, studied with anti-endothelial antibodies in chicken-quail chimeras. Circ Res 73: 559–568.
Limana F, Bertolami C, Mangoni A, et al: 2009. Myocardial infarction induces embryonic reprogramming of epicardial c-kit (+) cells: role of the pericardial fluid. JMCC 48:609–618. Limana F, Zacheo A, Mocini D, et al: 2007. Identification of myocardial and vascular precursor cells in human and mouse epicardium. Circ Res 101:1255–1265. Ma Q, Zhou B & Pu WT: 2008. Reassessment of Isl1 and Nkx2-5 cardiac fate maps using a Gata4-based reporter of Cre activity. Dev Biol 323:98–104. Manner J: 1999. Does the subepicardial mesenchyme contribute myocardioblasts to the myocardium of the chick embryo heart? A quail–chick chimera study tracing the fate of the epicardial primordium. Anat Rec 255: 212–226. Manner J, Perez-Pomares JM, Macias D, et al: 2001. The origin, formation and developmental significance of the epicardium: a review. Cells Tissues Organs 169:89–103. Martinez-Estrada OM, Lettice LA, Essafi A, et al: 2010. Wt1 is required form cardiovascular progenitor cell formation through transcriptional control of Snail and Ecadherin. Nat Genet 42:89–93. Mikawa T & Fischman DA: 1992. Retroviral analysis of cardiac morphogenesis: discon-
TCM Vol. 20, No. 1, 2010
Prall OWJ, Menon MK, Solloway MJ, et al: 2007. An Nkx2-5/Bmp2/Smad1 negative feedback loop controls heart progenitor specification and proliferation. Cell 128: 947–959. Rochais F, Mesbah K & Kelly RG: 2009. Signaling pathways controlling second heart field development. Circ Res 104: 933–942.
progenitor cells conferred by a 3′UTR-iresCre allele of the homeobox gene Nkx2-5. Int J Dev Bank 46:431–439. Van Den Berg G, Abu-Issa R, De Boer BA, et al: 2009. A caudal proliferating growth center contributes to both poles of the forming heart tube. Circ Res 104:179–188. Van Wijk B, Moorman AFM & Van Den Hoff MJB: 2007. Role of bone morphogenetic proteins in cardiac differentiation. Cardiovasc Res 74:244–255. Van Wijk B, Van Den Berg G, Abu-Issa R, et al: 2009. Epicardium and myocardium separate from a common precursor pool by crosstalk between bone morphogenetic protein- and fibroblast growth factor-signaling pathways. Circ Res 105:431–441. Wessels A & Perez-Pomares JM: 2004. The epicardium and epicardially derived cells (EPDCs) as cardiac stem cells. Anat Rec 276A:43–57. Wiese C, Grieskamp T, Airik R, et al: 2009. Formation of the sinus node head and differentiation of sinus node myocardium are independently regulated by Tbx18 and Tbx3. Circ Res 104:388–397.
Schlueter J & Brand T: 2009. A right-sided pathway involving FGF8/Snail controls asymmetric development of the proepicardium in the chick embryo. Proc Natl Acad Sci U S A 106:7485–7490.
Winter EM, Grauss RW, Hogers B, et al: 2007. Preservation of left ventricular function and attenuation of remodeling after transplantation of human epicardium-derived cells into the infarcted mouse heart. Circulation 116:917–927.
Schlueter J, Manner J & Brand T: 2006. BMP is an important regulator of proepicardial identity in the chick embryo. Dev Biol 295: 546–558.
Zhou B, Ma Q, Rajagopal S, et al: 2008a. Epicardial progenitors contribute to the cardiomyocyte lineage in the developing heart. Nature 454:109–113.
Schulte I, Schlueter J, Abu-Issa R, et al: 2007. Morphological and molecular left-right asymmetries in the development of the proepicardium: a comparative analysis on mouse and chick embryos. Dev Dyn 236: 684–695.
Zhou B, Von Gise A, Ma Q, et al: 2008b. Nkx25- and Isl1-expressing cardiac progenitors contribute to proepicardium. Biochem Biophys Res Commun 375:450–453.
Soriano P: 1999. Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet 21:70–71. Stanley EG, Biben C, Elefanty A, et al: 2002. Efficient Cre-mediated deletion in cardiac
PII S1050-1738(10)00029-0
TCM
7