Tbx18 regulates development of the epicardium and coronary vessels

The epicardium and coronary vessels originate from progenitor cells in the proepicardium. Here we show that Tbx18, a T-box family member highly expressed in the proepicardium, controls critical early steps in coronary development. In Tbx18^−/− mouse embryos, both the epicardium and coronary vessels exhibit structural and functional defects. At E12.5, the Tbx18-deficient epicardium contains protrusions and cyst-like structures overlying a disorganized coronary vascular plexus that contains ectopic structures resembling blood islands. At E13.5, the left and right coronary stems form correctly in mutant hearts. However, analysis of PECAM-1 whole mount immunostaining, distribution of SM22α^lacZ/+ activity, and analysis of coronary vascular casts suggest that defective vascular plexus remodeling produces a compromised arterial network at birth consisting of fewer distributing conduit arteries with smaller lumens and a reduced capacity to conduct blood flow. Gene expression profiles of Tbx18^−/− hearts at E12.5 reveal altered expression of 79 genes that are associated with development of the vascular system including sonic hedgehog signaling components patched and smoothened, VEGF-A, angiopoietin-1, endoglin, and Wnt factors compared to wild type hearts. Thus, formation of coronary vasculature is responsive to Tbx18-dependent gene targets in the epicardium, and a poorly structured network of coronary conduit vessels is formed in Tbx18 null hearts due to defects in epicardial cell signaling and fate during heart development. Lastly, we demonstrate that Tbx18 possesses a SRF/CArG box dependent repressor activity capable of inhibiting progenitor cell differentiation into smooth muscle cells, suggesting a potential function of Tbx18 in maintaining the progenitor status of epicardial-derived cells.     

Nf1 limits epicardial derivative expansion by regulating epithelial to mesenchymal transition and proliferation

The epicardium is the primary source of coronary vascular smooth muscle cells (cVSMCs) and fibroblasts that reside in the compact myocardium. To form these epicardial-derived cells (EPDCs), the epicardium undergoes the process of epithelial to mesenchymal transition (EMT). Although several signaling pathways have been identified that disrupt EMT, no pathway has been reported that restricts this developmental process. Here, we identify neurofibromin 1 (Nf1) as a key mediator of epicardial EMT. To determine the function of Nf1 during epicardial EMT and the formation of epicardial derivatives, cardiac fibroblasts and cVSMCs, we generated mice with a tissue-specific deletion of Nf1 in the epicardium. We found that mutant epicardial cells transitioned more readily to mesenchymal cells in vitro and in vivo. The mesothelial epicardium lost epithelial gene expression and became more invasive. Using lineage tracing of EPDCs, we found that the process of EMT occurred earlier in Nf1 mutant hearts, with an increase in epicardial cells entering the compact myocardium. Moreover, loss of Nf1 caused increased EPDC proliferation and resulted in more cardiac fibroblasts and cVSMCs. Finally, we were able to partially reverse the excessive EMT caused by loss of Nf1 by disrupting Pdgfrα expression in the epicardium. Conversely, Nf1 activation was able to inhibit PDGF-induced epicardial EMT. Our results demonstrate a regulatory role for Nf1 during epicardial EMT and provide insights into the susceptibility of patients with disrupted NF1 signaling to cardiovascular disease.     

BAF200 Is Required for Heart Morphogenesis and Coronary Artery Development

ATP-dependent SWI/SNF chromatin remodeling complexes utilize ATP hydrolysis to non-covalently change nucleosome-DNA interactions and are essential in stem cell development, organogenesis, and tumorigenesis. Biochemical studies show that SWI/SNF in mammalian cells can be divided into two subcomplexes BAF and PBAF based on the subunit composition. ARID2 or BAF200 has been defined as an intrinsic subunit of PBAF complex. However, the function of BAF200 in vivo is not clear. To dissect the possible role of BAF200 in regulating embryogenesis and organ development, we generated BAF200 mutant mice and found they were embryonic lethal. BAF200 mutant embryos exhibited multiple cardiac defects including thin myocardium, ventricular septum defect, common atrioventricular valve, and double outlet right ventricle around E14.5. Moreover, we also detected reduced intramyocardial coronary arteries in BAF200 mutants, suggesting that BAF200 is required for proper migration and differentiation of subepicardial venous cells into arterial endothelial cells. Our work revealed that PBAF complex plays a critical role in heart morphogenesis and coronary artery angiogenesis.     

Signaling via the Tgf-beta type I receptor Alk5 in heart development

Trophic factors secreted both from the endocardium and epicardium regulate appropriate growth of the myocardium during cardiac development. Epicardially-derived cells play also a key role in development of the coronary vasculature. This process involves transformation of epithelial (epicardial) cells to mesenchymal cells (EMT). Similarly, a subset of endocardial cells undergoes EMT to form the mesenchyme of endocardial cushions, which function as primordia for developing valves and septa. While it has been suggested that transforming growth factor-βs (Tgf-β) play an important role in induction of EMT in the avian epi- and endocardium, the function of Tgf-βs in corresponding mammalian tissues is still poorly understood. In this study, we have ablated the Tgf-β type I receptor Alk5 in endo-, myo- and epicardial lineages using the Tie2-Cre, Nkx2.5-Cre, and Gata5-Cre driver lines, respectively. We show that while Alk5-mediated signaling does not play a major role in the myocardium during mouse cardiac development, it is critically important in the endocardium for induction of EMT both in vitro and in vivo. Moreover, loss of epicardial Alk5-mediated signaling leads to disruption of cell-cell interactions between the epicardium and myocardium resulting in a thinned myocardium. Furthermore, epicardial cells lacking Alk5 fail to undergo Tgf-β-induced EMT in vitro. Late term mutant embryos lacking epicardial Alk5 display defective formation of a smooth muscle cell layer around coronary arteries, and aberrant formation of capillary vessels in the myocardium suggesting that Alk5 is controlling vascular homeostasis during cardiogenesis. To conclude, Tgf-β signaling via Alk5 is not required in myocardial cells during mammalian cardiac development, but plays an irreplaceable cell-autonomous role regulating cellular communication, differentiation and proliferation in endocardial and epicardial cells.     

Signaling Pathways That Control Rho Kinase Activity Maintain the Embryonic Epicardial Progenitor State

This study identifies signaling pathways that play key roles in the formation and maintenance of epicardial cells, a source of progenitors for coronary smooth muscle cells (SMCs). After epithelial to mesenchymal transition (EMT), mesenchymal cells invade the myocardium to form coronary SMCs. RhoA/Rho kinase activity is required for EMT and for differentiation into coronary SMCs, whereas cAMP activity is known to inhibit EMT in epithelial cells by an unknown mechanism. We use outgrowth of epicardial cells from E9.5 isolated mouse proepicardium (PE) explants, wild type and Epac1 null E12.5 mouse heart explants, adult rat epicardial cells, and immortalized mouse embryonic epicardial cells as model systems to identify signaling pathways that regulate RhoA activity to maintain the epicardial progenitor state. We demonstrate that RhoA activity is suppressed in the epicardial progenitor state, that the cAMP-dependent Rap1 GTP exchange factor (GEF), Epac, known to down-regulate RhoA activity through activation of Rap1 GTPase activity increased, that Rap1 activity increased, and that expression of the RhoA antagonistic Rnd proteins known to activate p190RhoGAP increased and associated with p190RhoGAP. Finally, EMT is associated with increased p63RhoGEF and RhoGEF-H1 protein expression, increased GEF-H1 activity, with a trend in increased p63RhoGEF activity. EMT is suppressed by partial silencing of p63RhoGEF and GEF-H1. In conclusion, we have identified new signaling molecules that act together to control RhoA activity and play critical roles in the maintenance of coronary smooth muscle progenitor cells in the embryonic epicardium. We suggest that their eventual manipulation could promote revascularization after myocardial injury.     

FGFR-1 is required by epicardium-derived cells for myocardial invasion and correct coronary vascular lineage differentiation

Critical steps in coronary vascular formation include the epithelial-mesenchyme transition (EMT) that epicardial cells undergo to become sub-epicardial; the invasion of the myocardium; and the differentiation of coronary lineages. However, the factors controlling these processes are not completely understood. Epicardial and coronary vascular precursors migrate to the avascular heart tube during embryogenesis via the proepicardium (PE). Here, we show that in the quail embryo fibroblast growth factor receptor (FGFR)-1 is expressed in a spatially and temporally restricted manner in the PE and epicardium-derived cells, including vascular endothelial precursors, and is up-regulated in epicardial cells after EMT. We used replication-defective retroviral vectors to over-express or knock-down FGFR-1 in the PE. FGFR-1 over-expression resulted in increased epicardial EMT. Knock-down of FGFR-1, however, did not inhibit epicardial EMT but greatly compromised the ability of PE progeny to invade the myocardium. The latter could, however, contribute to endothelia and smooth muscle of sub-epicardial vessels. Correct FGFR-1 levels were also important for correct coronary lineage differentiation with, at E12, an increase in the proportion of endothelial cells amongst FGFR-1 over-expressing PE progeny and a decrease in the proportion of smooth muscle cells in antisense FGFR-1 virus-infected PE progeny. Finally, in a heart explant system, constitutive activation of FGFR-1 signaling in epicardial cells resulted in increased delamination from the epicardium, invasion of the sub-epicardium, and invasion of the myocardium. These data reveal novel roles for FGFR-1 signaling in epicardial biology and coronary vascular lineage differentiation, and point to potential new therapeutic avenues.     

Epithelial-to-mesenchymal transformation alters electrical conductivity of human epicardial cells

The myocardium of the developing heart tube is covered by epicardium. These epicardial cells undergo a process of epithelial-to-mesenchymal transformation (EMT) and develop into epicardium-derived cells (EPDCs). The ingrowing EPDCs differentiate into several celltypes of which the cardiac fibroblasts form the main group. Disturbance of EMT of the epicardium leads to serious hypoplasia of the myocardium, abnormal coronary artery differentiation and Purkinje fibre paucity. Interestingly, the electrophysiological properties of epicardial cells and whether EMT influences electrical conductivity of epicardial cells is not yet known. We studied the electrophysiological aspects of epicardial cells before and after EMT in a dedicated in vitro model, using micro-electrode arrays to investigate electrical conduction across epicardial cells. Therefore, human adult epicardial cells were placed between two neonatal rat cardiomyocyte populations. Before EMT the epicardial cells have a cobblestone (epithelium-like) phenotype that was confirmed by staining for the cell-adhesion molecule β-catenin. After spontaneous EMT in vitro the EPDCs acquired a spindle-shaped morphology confirmed by vimentin staining. When comparing both types we observed that the electrical conduction is influenced by EMT, resulting in significantly reduced conductivity of spindle-shaped EPDCs, associated with a conduction block. Furthermore, the expression of both gap junction (connexins 40, Cx43 and Cx45) and ion channel proteins (SCN5a, CACNA1C and Kir2.1) was down-regulated after EMT. This study shows for the first time the conduction differences between epicardial cells before and after EMT. These differences may be of relevance for the role of EPDCs in cardiac development, and in EMT-related cardiac dysfunction.     

Epithelial‐to‐mesenchymal transition in epicardium is independent of snail1

The epicardium is the outer epithelial covering the heart. This tissue undergoes an epithelial‐to‐mesenchymal transition (EMT) to generate mesenchymal epicardial‐derived cells (EPDCs) that populate the extracellular matrix of the subepicardium and contribute to the development of the coronary vessels and cardiac interstitial cells. Although epicardial EMT plays a crucial role in heart development, the molecular regulation of this process is incompletely understood. Here we examined the possible role of the EMT regulator Snail1 in this process. Snail1 is expressed in the epicardium and EPDCs during mouse cardiac development. To determine the function of Snail1 in epicardial EMT, we deleted Snail1 in the epicardium using Wt1‐ and Tbx18‐Cre drivers. Unexpectedly, epicardial‐specific Snail1 mutants are viable and fertile and do not display any obvious morphological or functional cardiac abnormalities. Molecular analysis of these mice reveals that epicardial EMT occurs normally, and epicardial derivatives are established in these mutants. We conclude that Snail1 is not required for the initiation and progression of embryonic epicardial EMT. genesis 51:32–40, 2013. © 2012 Wiley Periodicals, Inc.     

VCAM-1 inhibits TGFbeta stimulated epithelial-mesenchymal transformation by modulating Rho activity and stabilizing intercellular adhesion in epicardial mesothelial cells

Regulation of epithelial-mesenchymal transformation (EMT) is of central importance both in normal development and in disease. During heart development, cells of the superficial epicardial mesothelium undergo EMT to give rise to precursor cells of the coronary vasculature and cardiac fibroblasts. Here we report that the alpha(4)beta(1) integrin ligand, VCAM-1, inhibits EMT of chick epicardial mesothelial cells stimulated by TGFbeta isoforms. We further investigated the molecular basis of this inhibition using cultured chick embryonic and rat adult epicardial mesothelial cells. We observed that VCAM-1 increased cortical actin filaments at intercellular junctions and reduced stress fibers across epicardial cells. VCAM-1 inhibited stress fiber formation by TGFbeta1, TGFbeta2, TGFbeta3 and lysophosphatidic acid and altered Rho activity stimulated by TGFbeta3. This was accompanied by an increase in tyrosine phosphorylation of p190RhoGAP. All three TGFbeta isoforms weakened intercellular adhesion, reduced membrane localization of beta-catenin and E-cadherin and stimulated epicardial EMT in chick hearts. Each of these effects was restricted by simultaneous VCAM-1 treatment. Our data support the hypothesis that VCAM-1 can alter epicardial EMT at two key points: it limits Rho-dependent events such as stress fiber formation and it maintains the association of beta-catenin and E-cadherin with the adherens junction.     

BMP2 signals loss of epithelial character in epicardial cells but requires the Type III TGFβ receptor to promote invasion

Coronary vessel development depends on a subpopulation of epicardial cells that undergo epithelial to mesenchymal transformation (EMT) and invade the subepicardial space and myocardium. These cells form the smooth muscle of the vessels and fibroblasts, but the mechanisms that regulate these processes are poorly understood. Mice lacking the Type III Transforming Growth Factor β Receptor (TGFβR3) die by E14.5 due to failed coronary vessel development accompanied by reduced epicardial cell invasion. BMP2 signals via TGFβR3 emphasizing the importance of determining the relative contributions of the canonical BMP signaling pathway and TGFβR3-dependent signaling to BMP2 responsiveness. Here we examined the role of TGFβR3 in BMP2 signaling in epicardial cells. Whereas TGFβ induced loss of epithelial character and smooth muscle differentiation, BMP2 induced an ALK3-dependent loss of epithelial character and modestly inhibited TGFβ-stimulated differentiation. Tgfbr3(-/-) cells respond to BMP2 indicating that TGFβR3 is not required. However, Tgfbr3(-/-) cells show decreased invasion in response to BMP2 and overexpression of TGFβR3 in Tgfbr3(-/-) cells rescued invasion. Invasion was dependent on ALK5, ALK2, ALK3, and Smad4. Expression of TGFβR3 lacking the 3 C-terminal amino acids required to interact with the scaffolding protein GIPC (GAIP-interacting protein, C terminus) did not rescue. Knockdown of GIPC in Tgfbr3(+/+) or Tgfbr3(-/-) cells rescued with TGFβR3 decreased BMP2-stimulated invasion confirming a requirement for TGFβR3/GIPC interaction. Our results reveal the relative roles of TGFβR3-dependent and TGFβR3-independent signaling in the actions of BMP2 on epicardial cell behavior and demonstrate the critical role of TGFβR3 in mediating BMP2-stimulated invasion.     

The cytoplasmic domain of TGFβR3 through its interaction with the scaffolding protein, GIPC, directs epicardial cell behavior

The epicardium is a major contributor of the cells that are required for the formation of coronary vessels. Mice lacking both copies of the gene encoding the Type III Transforming Growth Factor β Receptor (TGFβR3) fail to form the coronary vasculature, but the molecular mechanism by which TGFβR3 signals coronary vessel formation is unknown. We used intact embryos and epicardial cells from E11.5 mouse embryos to reveal the mechanisms by which TGFβR3 signals and regulates epicardial cell behavior. Analysis of E13.5 embryos reveals a lower rate of epicardial cell proliferation and decreased epicardially derived cell invasion in Tgfbr3^−/− hearts. Tgfbr3^−/− epicardial cells in vitro show decreased proliferation and decreased invasion in response to TGFβ1 and TGFβ2. Unexpectedly, loss of TGFβR3 also decreases responsiveness to two other important regulators of epicardial cell behavior, FGF2 and HMW-HA. Restoring full length TGFβR3 in Tgfbr3^−/− cells rescued deficits in invasion in vitro in response TGFβ1 and TGFβ2 as well as FGF2 and HMW-HA. Expression of TGFβR3 missing the 3 C-terminal amino acids that are required to interact with the scaffolding protein GIPC1 did not rescue any of the deficits. Overexpression of GIPC1 alone in Tgfbr3^−/− cells did not rescue invasion whereas knockdown of GIPC1 in Tgfbr3^+/+ cells decreased invasion in response to TGFβ2, FGF2, and HMW-HA. We conclude that TGFβR3 interaction with GIPC1 is critical for regulating invasion and growth factor responsiveness in epicardial cells and that dysregulation of epicardial cell proliferation and invasion contributes to failed coronary vessel development in Tgfbr3^−/− mice.     

Immunofluorescent studies on titin and myosin in developing hearts of normal and cardiac mutant axolotls

Homozygous recessive cardiac mutant gene c in the axolotl, Ambystoma mexicanum, results in a failure of the embryonic heart to initiate beating. Previous studies show that mutant axolotl hearts fail to form sarcomeric myofibrils even though hearts from their normal siblings exhibit organized myofibrils beginning at stage 34–35. In the present study, the proteins titin and myosin are studied using normal (+/+) axolotl embryonic hearts at stages 26–35. Additionally, titin is examined in normal (+/c) and cardiac mutant (c/c) embryonic axolotl hearts using immunofluorescent microscopy at stages 35–42. At tailbud stage-26, the ventromedially migrating sheets of precardiac mesoderm appear as two-cell-layers. Myosin shows periodic staining at the cell peripheries of the presumptive heart cells at this stage, whereas titin is not yet detectable by immunofluorescent microscopy. At preheartbeat stages 32–33, a myocardial tube begins to form around the endocardial tube. In some areas, periodic myosin staining is found to be separated from the titin staining; other areas in the heart at this stage show a co-localization of the two proteins. Both titin and myosin begin to incorporate into myofibrils at stage 35, when normal hearts initiate beating. Additionally, areas with amorphous staining for both proteins are observed at this stage. These observations indicate that titin and myosin accumulate independently at very early premyofibril stages; the two proteins then appear to associate closely just before assembly into myofibrils. Staining for titin in freshly frozen and paraffin-embedded tissues of normal embryonic hearts at stages 35, 39, and 41 reveals an increased organization of the protein into sarcomeres as development progresses. The mutant siblings, however, first show titin staining only limited to the peripheries of yolk platelets. Although substantial quantities of titin accumulate in mutant hearts at later stages of development (39 and 41), it does not become organized into myofibrils as in normal cells at these stages. © 1994 Wiley-Liss, Inc.     

The dynamic expression of tenascin-C and tenascin-X during early heart development in the mouse

One of a family of extracellular matrix proteins, tenascin-C (TNC) is expressed in a spatiotemporally restricted pattern associated with tissue remodeling during embryonic development, wound healing, cancer invasion and tissue regeneration. Another form, tenascin-X (TNX), is found in most tissues but most predominantly in heart and muscle, often complementarily to TNC. The present analysis demonstrated their expression during early heart development, using mouse lines containing the lacZ gene targeted to the TNC locus, by RT-PCR, immunohistochemistry, and in situ hybridization. TNC was transiently expressed at important steps during heart development: (1) precardiac mesodermal cells differentiating to cardiomyocytes and endocardial cells at E 7.5 - 8.5; (2) cardiomyocytes in the outflow tract at E 8.5 - 12; (3) endocardial cells forming cushion tissue at E 9.5 - 13; and (4) mesenchymal cells in the proepicardial organ (PEO), the precursors of coronary vessels, at E 9.5. When PEO cells were transferred onto the heart surface, the expression of TNC was downregulated, while TNX was upregulated at E 11. Initially, epicardial cells around the AV groove and atrium started to express TNX. TNX-positive cells then gradually spread all over the entire surface of the heart and invaded and formed primitive vascular channels in the myocardium. Despite restricted expression at important sites and steps during cardiogenesis, the hearts of TNC deficient mice developed normally. No difference in the expression pattern of TNX were observed in TNC knockout and wild mice. These results suggest; (1) TNC could play important roles in the differentiation of cardiomyocytes and the early morphogenesis of the heart; (2) TNX could be involved in coronary vasculogenesis; (3) TNX does not compensate for the loss of TNC.     

Embryonic development is disrupted by modest increases in vascular endothelial growth factor gene expression

Previous work has shown that heterozygocity for a null mutation of the VEGF-A gene, resulting in a 50% reduction in VEGF-A expression, is embryonic lethal at embroyonic day (E) 9.5 in mice. We now show that two- to threefold overexpression of VEGF-A from its endogenous locus results in severe abnormalities in heart development and embryonic lethality at E12.5-E14. The mutant embryos displayed an attenuated compact layer of myocardium, overproduction of trabeculae, defective ventricular septation and abnormalities in remodeling of the outflow track of the heart. In addition, aberrant coronary development was characterized by formation of oversized epicardial vessels, apparently through vasculogenesis. We infer that embryonic survival requires a narrow window of VEGF-A expression.     

Relationship between cardiac protein tyrosine phosphorylation and myofibrillogenesis during axolotl heart development

The axolotl, Ambystoma mexicanum, is a useful system for studying embryogenesis and cardiogenesis. To understand the role of protein tyrosine phosphorylation during heart development in normal and cardiac mutant axolotl embryonic hearts, we have investigated the state of protein tyrosine residues (phosphotyrosine, P-Tyr) and the relationship between P-Tyr and the development of organized sarcomeric myofibrils by using confocal microscopy, two-dimensional isoelectric focusing (IEF)/SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblotting analyses. Western blot analyses of normal embryonic hearts indicate that several proteins were significantly tyrosine phosphorylated after the initial heartbeat stage (stage 35). Mutant hearts at stages 40-41 showed less tyrosine phosphorylated staining as compared to the normal group. Two-dimensional gel electrophoresis revealed that most of the proteins from mutant hearts had a lower content of phosphorylated amino acids. Confocal microscopy of stage 35 normal hearts using phosphotyrosine monoclonal antibodies demonstrated that P-Tyr staining gradually increased being localized primarily at cell-cell boundaries and cell-extracellular matrix boundaries. In contrast, mutant embryonic hearts showed a marked decrease in the level of P-Tyr staining, especially at sites of cell-cell and cell-matrix junctions. We also delivered an anti-phosphotyrosine antibody (PY 20) into normal hearts by using a liposome-mediated delivery method, which resulted in a disruption of the existing cardiac myofibrils and reduced heartbeat rates. Our results suggest that protein tyrosine phosphorylation is critical during myofibrillogenesis and embryonic heart development in axolotls.     

Normal patterning of the coronary capillary plexus is dependent on the correct transmural gradient of FGF expression in the myocardium

The formation of the coronary vessel system is vital for heart development, an essential step of which is the establishment of a capillary plexus that displays a density gradient across the myocardial wall, being higher on the epicardial than the endocardial side. This gradient in capillary plexus formation develops concurrently with transmural gradients of myocardium-derived growth factors, including FGFs. To test the role of the FGF expression gradient in patterning the nascent capillary plexus, an ectopic FGF-over-expressing site was created in the ventricular myocardial wall in the quail embryo via retroviral infection from E2-2.5, thus abolishing the transmural gradient of FGFs. In FGF virus-infected regions of the ventricular myocardium, the capillary density across the transmural axis shifted away from that in control hearts at E7. This FGF-induced change in vessel patterning was more profound at E12, with the middle zone becoming the most vascularized. An up-regulation of FGFR-1 and VEGFR-2 in epicardial and subepicardial cells adjacent to FGF virus-infected myocardium was also detected, indicating a paracrine effect on induction of vascular signaling components in coronary precursors. These results suggest that correct transmural patterning of coronary vessels requires the correct transmural expression of FGF and, therefore, FGF may act as a template for coronary vessel patterning.