Initial formation of the epicardium, epicardial EMT, and EPDC lineage determination are regulated by a complex network of transcription factors, including the zinc finger transcription factors Wt1, Snai1, and Snai2, as well as the bHLH transcription factors Tcf21, Scleraxis, Twist1, and Hand2 (
Figure 1) [
8,
9,
10,
11]. Additional factors, including Tbx18, Nfatc1, Sox9, and C/EBP, regulate aspects of EPDC lineage development. Signaling pathways and transcription factors together regulate EPDC behavior and differentiation into cardiac fibroblasts and vascular SMCs [
4]. Transcription factors expressed in EPDCs, including Wt1, Tbx18, Tcf21, Snai1, and C/EBP, are reactivated in cardiac injury and may mark progenitor or reparative populations in the disease state [
18,
31,
32].
Figure 1.
Schematic depicting transcription factor regulation of epicardial cells during embryonic heart development. Several transcription factors are expressed during epicardial epithelial-to-mesenchymal transition (EMT), epicardium-derived cell (EPDC) lineage specification, and EPDC differentiation into vascular smooth muscle cells and cardiac fibroblasts. See text for details and references.
3.1. Wt1
The zinc finger transcription factor Wt1 was originally described as a tumor suppressor gene that is mutated in Wilms’ tumor patients [
33]. Wt1 is robustly expressed in the septum transversum/ pericardial mesothelium, the PE, and the epicardium [
34,
35]. Following epicardial EMT, Wt1 expression is rapidly downregulated in invading EPDCs in the developing heart [
36]. Therefore, Wt1 is expressed in EPDC progenitors with expression that diminishes prior to EPDC differentiation. Mice lacking Wt1 have epicardial defects with a paucity of EPDCs, suggesting an EMT defect [
12,
37,
38]. Wt1 is necessary and sufficient to activate transcription of
α4integrin (Itga4) via the proximal promoter (
Table 1), and
Itga4 is required to maintain epicardial adhesion and integrity [
11]. In addition, Wt1 directly regulates
Snai1 and
Snai2 (Slug) transcription in the epicardium [
39,
40]. Therefore, Wt1 is a crucial component of the mechanism regulating epicardial adhesion and EMT. Wt1 is required to promote epicardial expression of additional downstream targets, including
Nestin, a component of intermediate filaments,
TrkB (Tyrosine kinase type B receptor), important for BDNF (brain-derived neurotrophic factor) signaling and vascularization, and
Coronin1B, which is crucial for cell motility [
41,
42,
43]. Thus, loss of Wt1 adversely affects the cytoskeleton, thereby impacting EMT.
Table 1.
Transcription factor expression and function in epicardial development (see text for details and references).
Table 1.
Transcription factor expression and function in epicardial development (see text for details and references).
Gene | Loss-of-function cardiac phenotype a | Known downstream targets expressed in EPDCs | References |
---|
Wt1 | Ventricular non-compaction; impaired epicardial EMT; impaired coronary plexus formation; pericardial hemorrhaging; die by E13.5 | Itga4, Nestin, TrkB, Coronin1B, Raldh2, Snai1, Snai2 | [11,12,37,38,39,40,41,42,43] |
Tbx18 | Caval vein defects; sinus horn myocardial hypoplasia; neonatal lethality | Snai2 | [40,74,75] |
Tcf21 | Aberrant smooth muscle differentiation; loss of cardiac fibroblasts; pericardial hemorrhaging; neonatal lethality | None identified | [8,13,57] |
Nfatc1 | bReduced cardiac fibrous matrix with decreased coronary vessel penetration; neonatal lethality | Ctsk | [9,87,88] |
Snai1 | b,cPhenotypically normal and viable | E-cadherin, Mmp15 | [92,93,96,97] |
Snai2 | Phenotypically normal and viable | None identified | [40] |
Sox9 | Hypoplastic endocardial cushions. Embryonic lethality at E11.5-E12 due to congestive heart failure. | None identified | [102,104,108] |
Scleraxis | Thickened valves; viable | Col1a2 | [106,107] |
C/EBP | dImproved cardiac function after ischemia/reperfusion injury | Raldh2, Wt1 | [32] |
Hand2 | eEpicardial blistering; abnormal coronary vessel development; loss of cardiac fibroblasts; persistent truncus arteriosus. Embryonic lethality by E14.5. | Pdgfra | [101] |
Twist1 | Abnormal outflow tract endocardial cushion mesenchyme. Embryonic lethality by E11.5. | Tbx20, Snai2 | [30,76,100] |
Multiple signaling pathways required for EPDC lineage development are affected with loss of Wt1. Retinoic acid (RA) signaling is required during cardiac morphogenesis [
44]. Retinoid X Receptor α, which binds RA in the nucleus, is required during cardiac development, as
Rxrα null mice are embryonic lethal by E15 with ventricular hypoplasia and delayed formation of the epicardium [
45,
46,
47]. Wt1-deficient embryos have decreased expression of
Retinaldehyde dehydrogenase-2 (Raldh2), a direct downstream target of Wt1, and epicardial EMT is partially rescued by RA supplementation in Wt1-deficient embryos [
12,
37]. Interestingly, RA induces
Wt1 expression in proepicardial cells and EPDCs in cell culture supporting a feedforward regulatory mechanism [
8]. Canonical Wnt/β-Catenin signaling, required for epicardial EMT, ventricular compaction, and formation of the coronary plexus in mouse embryonic hearts, also is downstream of Wt1 [
12,
48,
49]. In Wt1 null embryos, the epicardium fails to undergo EMT and Wnt signaling is reduced [
12,
48,
49]. Therefore, Wt1 is a master regulator upstream of crucial signaling pathways, including Wnt/β-Catenin and RA, in epicardial development. In addition, Wt1, Wnt/β-Catenin, and Raldh2 are reactivated in mouse models of adult heart disease, including MI, ischemia/reperfusion (I/R), and pressure overload (
Figure 2) [
16,
18,
31,
50].
Initial Wt1Cre-based lineage studies reported that the majority of Wt1-derived cells differentiate into SM, but that some Wt1-derived cells differentiate into cardiomyocytes and endothelial cells [
7]. Wt1 lineage-derived cells also contribute to fibroblasts of the annulus fibrosis, interstitial fibroblasts, and AV valve parietal leaflet interstitial cells [
24,
30]. Very few, if any, endothelial cells are derived from the Wt1 lineage in these analyses [
7,
24,
30]. The report that Wt1 lineage-positive cells become cardiomyocytes, thereby supporting an epicardial origin for cardiac muscle, is controversial [
51,
52]. Caveats to this approach are that Wt1 expression is not completely epicardial-specific in addition to potential leakiness of Cre expression and inefficiency of recombination inherent to the Wt1Cre mouse lines [
51,
52]. Tamoxifen-inducible Wt1Cre lines add temporal and spatial specificity, but inefficient and variable recombination following tamoxifen induction is a concern with the Wt1CreERT2 mouse line [
51,
52]. It remains controversial whether small subpopulations of Wt1 lineage-positive epicardial cells become cardiomyocytes or endothelial cells. However, there is general agreement that the majority of Wt1Cre-positive epicardial derivatives become fibroblasts and vascular SMCs [
7,
24,
51].
3.2. Tcf21
The bHLH transcription factor Tcf21 (Pod1/Epicardin/Capsulin) is expressed in developing mesothelial cell populations, including the PE and epicardium, as well as kidney, lung, and reproductive tract [
53,
54,
55]. Loss of Tcf21 leads to kidney and lung defects, spleen agenesis, and neonatal lethality [
56,
57]. In the heart, Tcf21 is required for normal epicardial development and regulates EPDC differentiation into SM and fibroblast lineages [
8,
13]. Tcf21 deficiency leads to aberrant SM differentiation in the subepicardial mesenchyme and a paucity of cardiac fibroblasts in the myocardial interstitium [
8]. Expression of Tcf21, like Wt1, is induced by RA signaling in EPDCs, and RA inhibits SM differentiation of PE derivatives [
8,
58]. Tcf21 expression is downregulated in differentiated vascular SM in the myocardial interstitium, consistent with a repressive role in the differentiation of this lineage. Thus, Tcf21 and RA signaling together inhibit SM gene expression and differentiation in EPDC progenitor cells prior to their localization in the coronary vasculature. In contrast, Tcf21 expression promotes cardiac fibroblast identity and persists in differentiated cardiac interstitial and adventitial fibroblasts in the postnatal and adult heart [
8,
13,
59].
Figure 2.
Model depicting epicardial cell reactivation and expression of transcription factors, including Tcf21, Wt1, Tbx18, Snai1, and C/EBPβ, following myocardial infarction (MI) in the adult heart. Activated epicardial cells undergo EMT and invade the subepicardial space following MI. The ultimate fate of activated EPDCs and their ability to invade the myocardium in the infarcted heart has not yet been fully characterized. In the area of the infarct scar Tcf21, Wt1, Tbx18, and Scleraxis (Scx) also are expressed, and immune cells are present in the activated epicardium and fibrotic scar. Currently, it has not been reported whether epicardial transcription factors are activated in other forms of cardiac fibrosis. See text for details and references.
Figure 2.
Model depicting epicardial cell reactivation and expression of transcription factors, including Tcf21, Wt1, Tbx18, Snai1, and C/EBPβ, following myocardial infarction (MI) in the adult heart. Activated epicardial cells undergo EMT and invade the subepicardial space following MI. The ultimate fate of activated EPDCs and their ability to invade the myocardium in the infarcted heart has not yet been fully characterized. In the area of the infarct scar Tcf21, Wt1, Tbx18, and Scleraxis (Scx) also are expressed, and immune cells are present in the activated epicardium and fibrotic scar. Currently, it has not been reported whether epicardial transcription factors are activated in other forms of cardiac fibrosis. See text for details and references.
Tcf21 heterodimerizes with the class I bHLH transcription factor E12 [
60,
61]. Together, Tcf21 and E12 negatively regulate transcription [
60,
62]. Analysis of
Xenopus embryos indicates that Tcf21 functions as a transcriptional repressor with other repressor proteins to regulate PE-specific gene expression [
63]. Additional bHLH dimerization partners for Tcf21 have not been described, nor have Tcf21 downstream targets been identified in the heart
in vivo. Studies using a mesenchymal cell line derived from adult mouse kidney determined that Tcf21 binds to E-box DNA consensus sequences (CAnnTG) in the
SM22α,
Calponin, and
αSMA promoters [
64]. Overexpression of Tcf21 alone leads to decreased expression of SM22α, Calponin, and αSMA protein, whereas overexpression of Tcf21 and E2A results in increased SM22α, Calponin, and αSMA protein expression [
64]. Therefore, expression of
E2A, which encodes the E12 and E47 transcription factors, may influence the role of Tcf21 in terms of SM and myofibroblast downstream targets [
65]. In addition to acting as a transcriptional repressor, Tcf21 contains an activation domain at its C-terminus [
66,
67]. While expression of SM22α, Calponin, and αSMA is increased in Tcf21 null hearts [
8], direct regulatory interactions of Tcf21 with these gene regulatory elements have not yet been established in EPDCs. The dynamic and differential mechanisms by which Tcf21 regulates cell fate have yet to be determined. Likewise, the identity of Tcf21 E-box binding partners is likely to influence Tcf21 function in different contexts [
62].
Fate mapping studies with the tamoxifen-inducible Tcf21iCre mouse line demonstrate that Tcf21iCre-derived cells contribute to fibroblasts of the coronary adventitia and myocardial interstitium, in addition to coronary vascular SMCs, when Cre activity is induced during embryogenesis [
59]. In addition, Tcf21iCre-derived cells are detected in the gonads, lung, spleen, adrenal gland, and facial skeletal muscles [
59]. Interestingly, in the heart, lung, kidney, spleen, adrenal gland, testes, and ovaries, Tcf21iCre-derived cells contribute to interstitial cells that support organ function [
59]. Therefore, Tcf21 regulation of interstitial fibroblast formation may be conserved throughout the developing embryo. In the heart, fate mapping of the embryonic Tcf21 lineage marks fibroblasts and SMCs, but not cardiomyocytes or endothelial cells [
59,
68]. Postnatal induction of Tcf21iCre activity leads to recombination in cardiac interstitial cells, but not endothelial cells, supporting a homeostatic role for Tcf21 in fibroblast lineages after birth [
59]. As determined by genome-wide association studies of human coronary artery disease (CAD), a variant of TCF21 is associated with increased risk of CAD in European and Chinese Han populations [
69,
70]. Likewise, TCF21 is expressed in human cardiac fibrotic disease and ischemic cardiomyopathy ([
71]; Braitsch, unpublished). In addition, Tcf21 is reactivated following myocardial injury in adult mouse and zebrafish models (
Figure 2) [
15,
16,
18,
31,
68]. Therefore, Tcf21 is likely to play an important role in adult cardiac homeostasis and disease.
3.3. Tbx18
Tbx18, a member of the T-box transcription factor family, is expressed in the PE, epicardium, somites, limb buds, and genital ridge [
72,
73]. Mice lacking Tbx18 die at birth due to cyanosis resulting from severe defects of the axial skeleton [
10,
74]. In the heart, Tbx18 contributes to, and is required for, formation of the sinus horn myocardium at the venous pole of the heart [
75]. Loss of Tbx18 does not appear to affect epicardial development, as EPDCs are apparently unaffected in the
Tbx18−/− mouse heart [
74]. It is possible that Tbx20
, which is expressed in the epicardium and subepicardial EPDCs, may have overlapping or redundant functions with Tbx18 in these cells [
76]. Lineage-tracing analysis of a Tbx18Cre knock-in allele indicates that cells from the Tbx18Cre lineage differentiate into fibroblasts, vascular SMCs, and cardiomyocytes [
3]. However, Tbx18 is actively expressed in myocardium of the interventricular septum and left ventricle during mouse embryogenesis from E10.5-E16.5, supporting a nonepicardial source for Tbx18 lineage-positive cardiomyocytes [
75,
77]. In contrast, studies by multiple groups confirm that vascular SM and cardiac fibroblasts, but not endothelial cells, arise from a Tbx18-positive epicardial lineage [
3,
77,
78].
T-box transcription factors can act as transcriptional activators and/or repressors [
73]. In the developing somites, Tbx18 maintains anterior somite identity by acting as a transcriptional repressor of
Delta-like 1 (
Dll1), a Notch effector [
79]. In EPDCs, there is evidence that Tbx18 functions as a transcriptional repressor of SM differentiation, since ectopic expression of a transcriptional activator Tbx18VP16 leads to premature SM differentiation in the epicardium [
10]. Tbx18VP16-mediated SM differentiation in epicardial cells is reversed by Notch inhibition
in vitro [
10]. However, few cardiac-specific downstream targets of Tbx18 have been identified. Tbx18 directly binds and promotes epicardial
Snai2 expression, thereby promoting epicardial EMT in cell culture [
40]. Together these studies indicate that Tbx18 maintains progenitor cell identity by acting as a transcriptional repressor during embryonic development, often upstream of Notch signaling. In addition, Tbx18 is reactivated in epicardial cells in adult ischemic heart disease (
Figure 2) [
16,
31].
3.4. Nfatc1
Nfatc1 is a member of the nuclear factor of activated T cells family of transcription factors, which are activated and localized to the nucleus by Ca
2+ signaling via the calcium-responsive phosphatase calcineurin [
80]. Loss of Nfatc1 in mice leads to lethality at E12.5-E14.5 with defects in heart valve remodeling [
81,
82]. Nfatc1 is expressed in the endocardial cushions and remodeling heart valves, as well as in the PE, epicardium, and EPDCs during heart development [
9,
83,
84]. In the developing valves, Nfatc1 is required to promote endocardial cushion proliferation through the VEGF pathway and to regulate heart valve remodeling via Receptor Activator of Nuclear factor Kappa-B Ligand (RANKL) signaling [
83,
85,
86]. In EPDCs, Nfatc1 is required for invasion of the myocardium, and mice deficient in epicardial Nfatc1 have decreased interstitial fibrous matrix deposition and exhibit neonatal lethality [
9]. Likewise, epicardial loss of Nfatc1 results in decreased coronary vessel penetration, without affected SM differentiation, in the embryonic mouse heart [
9]. Specifically, epicardial Nfatc1 is necessary for RANKL promotion of
CathepsinK (Ctsk) mediated EPDC invasion of the myocardium [
9].
Ctsk is an ECM remodeling enzyme that facilitates cell migration and is a transcriptional target of Nfatc1 (
Table 1), first defined in osteoclast cell lineages [
87,
88]. Therefore, Nfatc1 is required for
Ctsk expression and cell invasion, necessary for EPDC lineage development, in a mechanism that also is active in developing osteoclasts and remodeling heart valves.
Nfatc1 also has been implicated in coronary endothelial lineage development. Nfatc1 is expressed in differentiated coronary endothelial cells, and calcineurin/NFAT signaling is required for coronary angiogenesis during embryonic heart development [
9,
89]. Targeted deletion of Nfatc1 with Wt1Cre or Gata5Cre does not prevent differentiation of coronary endothelial cells, but these Cre lines are not generally considered to be active in the endothelial lineage [
9,
51]. However, calcineurin/NFAT signaling is required in endothelial cells for coronary vessel development and is induced by VEGF signaling [
89]. Recently, endocardial endothelial cells were reported to be a source of coronary endothelial cells based on restriction of Nfatc1 expression to the endocardium [
27]. While these data do not take into account Nfatc1 expression in the epicardium and its necessity for EPDC invasion, they do support multiple sources of coronary endothelial cells that warrant further investigation [
6,
9,
27]. In addition, the specific functions and downstream targets of Nfatc1 in coronary endothelial cell differentiation have not been identified.
3.5. Snai1 and Snal2
The zinc finger transcription factors Snai1 (Snail1) and Snai2 (Snail2, Slug) are robustly expressed in the epicardium and EPDCs of mouse and chick embryonic hearts [
90,
91]. Snai1 promotes EMT in the endocardial cushions as well as in other organ systems and during tumorigenesis, in part via the Snai1 downstream target
Mmp15 [
92,
93]. However, there is conflicting evidence for the requirement for Snai1 and Snai2 in epicardial EMT [
39,
90,
94]. In cultured avian epicardial cells, Snai1 overexpression promotes cell migration and invasion [
94]. Similarly in mouse epicardial cell cultures, loss of Snai2 inhibits EMT, and
Snai2 gene expression is dependent on Wt1 and Tbx18 [
40]. Deletion of Wt1 from the more broadly expressed Gata5Cre lineage leads to loss of
Snai1 expression with concomitant epicardial EMT defects with embryonic lethality [
39,
95]. In contrast,
in vivo loss of Snai1 in Wt1Cre or Tbx18Cre lineages does not affect epicardial EMT or differentiation [
90]. In a variety of cell types, Snai1 represses expression of
E-cadherin and other adhesion molecules, which are required to maintain epithelial integrity [
96,
97]. Wt1Cre-mediated loss of the Notch pathway transcriptional activator
Rbpj leads to decreased expression of
Snai1, consistent with an observed EMT defect, as well as aberrant coronary SM differentiation [
98]. Together these studies suggest that Notch signaling regulates Snai1 and E-cadherin, both of which affect epicardial EMT. This same regulatory hierarchy also is active in endocardial cushion EMT [
99]. In addition,
Snai1 expression is reactivated in the infarct scar following MI (
Figure 2) [
31].
3.6. Twist1 and Hand2
In addition to Tcf21, the bHLH transcription factors Twist1 and Hand2 also have been implicated in EPDC development. Twist1 is expressed in EPDCs of avian embryos at the same time it is expressed in endocardial cushions, where it promotes mesenchymal cell proliferation and migration [
76]. In mice, EPDCs isolated from mouse AV canals express Twist1, in addition to Snai1, Snai2, and Smad1 markers of EMT [
30]. However, a specific function for Twist1 in the epicardium or EPDCs has not yet been demonstrated. In endocardial cushions, Twist1 promotes expression of genes associated with cell proliferation and migration, and a similar regulatory mechanism may be active in EPDCs [
76,
100]. For example,
Tbx20 is expressed in EPDCs as well as endocardial cushions and is a direct downstream target of Twist1 in endocardial cushion cells [
76,
100]. Likewise, Hand1 is expressed at the venous pole of the heart and cells of the Hand1 lineage contribute to epicardial progenitors [
101]. In addition, loss of Hand2 in the Hand1 lineage leads to epicardial blistering, abnormal coronary vessel development, and loss of cardiac fibroblasts [
101]. Hand2 promotes expression of
Pdgfra, which is required for epicardial EMT and epicardium-derived cardiac fibroblasts [
101,
102]. Additional studies are necessary to define the specific functions and transcriptional targets of Twist1, Hand1, and Hand2 in epicardial lineage development.
3.7. Scleraxis and Sox9
Scleraxis (Scx) is a bHLH transcription factor originally reported to be important in tendon development, and it also functions in cell lineage diversification in heart valvulogenesis [
103,
104,
105]. Scx is expressed in a subdomain of the mouse PE, beginning at E9.5, and in the epicardium at E10.5 [
6]. In the PE, cells that express Scx do not express Wt1 or Tbx18, demonstrating heterogeneity of this progenitor population [
6]. ScxCre-derived cells contribute to coronary endothelial cells on the surface of the heart and also to cardiomyocytes in the LV. However, ScxCre-positive cells are rarely detected in SM at E12.5, in contrast to Wt1Cre or Tbx18Cre-derived cells, providing evidence for distinct compartments of proepicardial cells that give rise to endothelial versus fibroblast and SM lineages. The specific function(s) of Scx in epicardial development has not been demonstrated, although loss of Scx leads to persistent expression of EMT markers and heart valve remodeling defects at E17.5 in mice [
106]. Interestingly, in adult
Scx−/− mice, thickening and increased collagen deposition are apparent in the AV annulus and mitral valve parietal leaflet that are derived from epicardium [
24,
30,
106]. In adult cardiac fibroblasts, Scx directly regulates
Col1a2 gene expression, and Scx expression also is induced after MI, supporting a role in cardiac fibrosis (
Figure 2) [
107]. However, additional studies are necessary to define the specific functions of Scx in epicardium-derived cell lineage development or pathogenesis related to EPDCs.
Sox9 is an SRY-related transcription factor that is crucial for heart valve development [
104]. During valvulogenesis, Sox9 is required for endocardial cushion EMT, progenitor cell proliferation, and proteoglycan-rich cell lineage development [
104,
108]. Sox9 also is expressed in EPDCs and is sufficient to promote epicardial EMT and migration [
102]. Therefore, mechanisms regulating EMT and mesenchymal proliferation may be conserved in endocardial cushions and epicardium. However, little is known of Sox9 functions in EPDCs, and defective EPDC lineage development has not been reported in Sox9-deficient mouse embryos.