Subepicardial Mesenchyme Research Articles

Functional Heterogeneity within the Developing Zebrafish Epicardium

SummaryThe epicardium is essential during cardiac development, homeostasis, and repair, and yet fundamental insights into its underlying cell biology, notably epicardium formation, lineage heterogeneity, and functional cross-talk with other cell types in the heart, are currently lacking. In this study, we investigated epicardial heterogeneity and the functional diversity of discrete epicardial subpopulations in the developing zebrafish heart. Single-cell RNA sequencing uncovered three epicardial subpopulations with specific genetic programs and distinctive spatial distribution. Perturbation of unique gene signatures uncovered specific functions associated with each subpopulation and established epicardial roles in cell adhesion, migration, and chemotaxis as a mechanism for recruitment of leukocytes into the heart. Understanding which mechanisms epicardial cells employ to establish a functional epicardium and how they communicate with other cardiovascular cell types during development will bring us closer to repairing cellular relationships that are disrupted during cardiovascular disease.

In vitro avian pro‐epicardial cell spreading challenges the notion of how epicardial cells spread during cardiac morphogenesis (LB65)

In the developing heart, the epicardium is derived from the pro‐epicardial organ (PEO), which spreads over the cardiac tube (purportedly as an epithelium), and then undergoes what is believed to be an epithelial‐to‐mesenchymal transition (EMT) to generate the subepicardial mesenchyme. We are interested in the process of epicardial cell spreading and the mechanisms that regulate the epicardial EMT. To study these processes, we have developed fluorescent labeling techniques to visualize the PEO and the spreading epicardium using time‐lapse confocal imaging in intact hearts and on a planar substratum. When the PEO is labeled and then explanted before it begins to spread in vivo, we saw that each cell has numerous filopodia and lamellipodia around its borders. Fixed images of spreading PEO cells labeled in ovo confirm the presence of numerous filopodia and lamellipodia in the spreading epicardium in vivo. Although the PEO has been assumed to spread as an epithelium with the force being generated at the free edge, our live‐cell imaging suggests that each cell in the epicardium is generating tractional force. These results show that real‐time behavior studies are critical since morphology alone does not reveal behavior. Our observations challenge the notion of how the PEO‐derived cells spread over the cardiac tube during cardiac morphogenesis.Grant Funding Source: Supported by NIHT32OD011147 (JV‐M), UCD GSSP Fellowship (JV‐M), UCD GGCP Fellowship (JV‐M), IOS1146480 (CAE)

Cardiac Regeneration from Activated Epicardium

In contrast to lower vertebrates, the mammalian heart has a very limited regenerative capacity. Cardiomyocytes, lost after ischemia, are replaced by fibroblasts. Although the human heart is able to form new cardiomyocytes throughout its lifespan, the efficiency of this phenomenon is not enough to substitute sufficient myocardial mass after an infarction. In contrast, zebrafish hearts regenerate through epicardial activation and initiation of myocardial proliferation. With this study we obtain insights into the activation and cellular contribution of the mammalian epicardium in response to ischemia. In a mouse myocardial infarction model we analyzed the spatio-temporal changes in expression of embryonic epicardial, EMT, and stem cell markers and the contribution of cells of the Wt1-lineage to the infarcted area. Though the integrity of the epicardial layer overlaying the infarct is lost immediately after the induction of the ischemia, it was found to be regenerated at three days post infarction. In this regenerated epicardium, the embryonic gene program is transiently re-expressed as well as proliferation. Concomitant with this activation, Wt1-lineage positive subepicardial mesenchyme is formed until two weeks post-infarction. These mesenchymal cells replace the cardiomyocytes lost due to the ischemia and contribute to the fibroblast population, myofibroblasts and coronary endothelium in the infarct, and later also to the cardiomyocyte population. We show that in mice, as in lower vertebrates, an endogenous, epicardium-dependent regenerative response to injury is induced. Although this regenerative response leads to the formation of new cardiomyocytes, their number is insufficient in mice but sufficient in lower vertebrates to replace lost cardiomyocytes. These molecular and cellular analyses provide basic knowledge essential for investigations on the regeneration of the mammalian heart aiming at epicardium-derived cells.

Epicardial–Myocardial Signaling Directing Coronary Vasculogenesis

The establishment of the coronary circulation is critical for the development of the embryonic heart. Over the last several years, there has been tremendous progress in elucidating the pathways that control coronary development. Interestingly, many of the pathways that regulate the development of the coronary vasculature are distinct from those governing vasculogenesis in the rest of the embryo. It is becoming increasingly clear that coronary development depends on a complex communication between the epicardium, the subepicardial mesenchyme, and the myocardium mediated in part by secreted growth factors. This communication coordinates the growth of the myocardium with the formation of the coronary vasculature. This review summarizes our present understanding of the role of these growth factors in the regulation of coronary development. Continued progress in this field holds the potential to lead to novel therapeutics for the treatment of patients with coronary artery disease.

A migratory role for EphrinB ligands in avian epicardial mesothelial cells

Little is known about the molecules that mediate the attachment of proepicardial cells to the heart. Ephrins are cell surface ligands for Eph tyrosine kinase receptors, molecules known to play a role in cell adhesion and migration. Here, we detected EphrinB ligands in proepicardial and epicardial mesothelial cells (EMCs) using reverse transcriptase-polymerase chain reaction, immunoblotting, immunolocalization, and EphB-Fc binding. Aggregated EphB-Fc fragments clustered ephrinB1 ligands on living EMCs indicating that they are cell surface expressed. In vitro assays demonstrated that ephrinB ligands participate in EMC migration but not cell adhesion. Localization studies in hearts at Hamburger and Hamilton stage 30 and older revealed that ephrinB1 is expressed in the epicardium and subepicardial mesenchyme of the atrioventricular sulcus. EMCs treated with platelet-derived growth factor-BB expressed smooth muscle markers but not ephrinB1. Our study supports an early role for ephrinB ligands for migration of epicardial cells and a later role in perivascular fibroblasts of coronary vessels in the atrioventricular sulcus.

Coronary Vessel Development Is Dependent on the Type III Transforming Growth Factor β Receptor

Transforming growth factor (TGF)beta receptor III (TGFbetaR3), or beta-glycan, binds all 3 TGFbeta ligands and inhibin with high affinity but lacks the serine/threonine kinase domain found in the type I and type II receptors (TGFbetaR1, TGFbetaR2). TGFbetaR3 facilitates signaling via TGFbetaR1/TGFbetaR2 but also has been suggested to play a unique and nonredundant role in TGFbeta signaling. Targeted deletion of Tgfbr3 revealed a requirement for Tgfbr3 during development of the coronary vessels. Coronary vasculogenesis is significantly impaired in null mice, with few vessels evident and numerous, persistent blood islands found throughout the epicardium. Tgfbr3-null mice die at embryonic day 14.5, the time when functional coronary vasculature is required for embryo viability. However, in null mice nascent coronary vessels attach to the aorta, form 2 coronary ostia, and initiate smooth muscle recruitment by embryonic day 14. Analysis of earlier developmental stages revealed defects in the epicardium. At embryonic day 13.5, these defects include an irregular and hypercellular epicardium with abundant subepicardial mesenchyme and a thin compact zone myocardium. Tgfbr3-null mice also displayed other defects in coronary development, including dysmorphic and distended vessels along the atrioventricular groove and subepicardial hemorrhage. In null mice, vessels throughout the yolk sac and embryo form and recruit smooth muscle in a pattern indistinguishable from heterozygous or wild-type littermates. These data demonstrate a requirement for Tgfbr3 during coronary vessel development that is essential for embryonic viability.

Origin, Fate, and Function of Epicardium-Derived Cells (EPDCs) in Normal and Abnormal Cardiac Development

During heart development, cells of the primary and secondary heart field give rise to the myocardial component of the heart. The neural crest and epicardium provide the heart with a considerable amount of nonmyocardial cells that are indispensable for correct heart development. During the past 2 decades, the importance of epicardium-derived cells (EPDCs) in heart formation became increasingly clear. The epicardium is embryologically formed by the outgrowth of proepicardial cells over the naked heart tube. Following epithelial-mesenchymal transformation, EPDCs form the subepicardial mesenchyme and subsequently migrate into the myocardium, and differentiate into smooth muscle cells and fibroblasts. They contribute to the media of the coronary arteries, to the atrioventricular valves, and the fibrous heart skeleton. Furthermore, they are important for the myocardial architecture of the ventricular walls and for the induction of Purkinje fiber formation.Whereas the exact signaling cascades in EPDC migration and function still need to be elucidated, recent research has revealed several factors that are involved in EPDC migration and specialization, and in the cross-talk between EPDCs and other cells during heart development. Among these factors are the Ets transcription factors Ets-1 and Ets-2. New data obtained with lentiviral antisense constructs targeting Ets-1 and Ets-2 specifically in the epicardium indicate that both factors are independently involved in the migratory behavior of EPDCs. Ets-2 seems to be especially important for the migration of EPDCs into the myocardial wall, and to subendocardial positions in the atrioventricular cushions and the trabeculae.With respect to the clinical importance of correct EPDC development, the relation with coronary arteriogenesis has been noted well before. In this review, we also propose a role for EPDCs in cardiac looping, and emphasize their contribution to the development of the valves and myocardial architecture. Lastly, we focus on the congenital heart anomalies that might be caused primarily by an epicardial developmental defect.

The Type III Transforming Growth Factor β Receptor ( tgfbr3 ) is Required for Coronary Vasculogenesis and Embryonic Viability

Transforming Growth Factor Beta (TGFβ) Receptor III (TBRIII) or betaglycan binds all three TGFβ ligands and inhibin with high affinity but lacks the serine/threonine kinase domain found in the Type I and Type II receptors (TBRI, TBRII). TBRIII can facilitate signaling via TBRI/TBRII but also has been suggested to play a unique and nonredundant role in TGFβ signaling. Targeted deletion of the gene encoding mouse TBRIII, tgfbr3, revealed a unique requirement for tgfbr3 during development of the coronary vessels. tgfbr3 null mice die around 14.5 dpc with fewer PECAM-positive coronary vessels and an apparent absence of remodeled vessels while other vascular beds appeared intact. Despite the paucity of coronary vessels, both right and left coronary ostia formed along the aortic wall. Analysis of earlier developmental stages revealed profound defects in the interaction of the proepicardium (PE) with the myocardium and subsequent formation of the epicardium (EP). At 13.5 dpc these defects include the continued presence of the PE and PE-derived vesicles, an irregular and hypercellular EP with excess subepicardial mesenchyme and a thin compact zone myocardium. tgfbr3 null mice also displayed other defects in coronary development including dysmorphic and distended vessels along the AV groove, subepicardial hemorrhage and multiple, large, persistent blood islands. These data demonstrate a requirement for tgfbr3 during coronary vasculogenesis that is essential for embryonic viability. NIH HL67105; Vanderbilt MSTP 5 T32 GM07347

Inhibition of α4-integrin stimulates epicardial–mesenchymal transformation and alters migration and cell fate of epicardially derived mesenchyme

Epithelial–mesenchymal transformation of the embryonic epicardium produces the subepicardial mesenchyme that is essential for normal coronary vascular development. Gene targeting experiments in mice have demonstrated an essential role for α4-integrin in normal epicardial development, but the precise cellular consequences of α4-integrin loss remain uncertain. To better understand the function of α4-integrin in epicardial development, we constructed a replication-incompetent adenovirus ( AdlacZα4AS) that expresses antisense chicken α4-integrin as the 3′ untranslated region of a lacZ reporter gene. This construct effectively labeled cells while greatly reducing levels of α4-integrin mRNA and protein. In quail chick chimeras, transplanted epicardial cells infected with AdlacZα4AS adhered to the heart and were incorporated into the epicardium, but 4 days after grafting, were largely absent from the epicardial epithelium, recapitulating the defect in α4-null mice. This did not result from epicardial cell apoptosis or anomalous migration of epicardial cells to extracardiac sites. Rather, AdlacZα4AS-infected epicardial cells were particularly invasive, being three to four times more likely to migrate to the interstitium of the myocardium than AdlacZ-infected epicardial cells. Accelerated epicardial–mesenchymal transformation and migration of α4-negative epicardium was observed in an organ culture system that does not require prior culture of epicardial cells. Remarkably, AdlacZα4AS infection also prevented targeting of epicardially derived mesenchyme to the media of developing coronary vasculature in the myocardial interstitium. This study provides evidence that epicardial α4-integrin normally restrains epicardial–mesenchymal transformation, invasion, and migration and is essential for correct targeting of epicardially derived mesenchyme to the developing coronary vasculature.

The contribution of the proepicardium to avian cardiovascular development

The embryonic epicardial layer of the heart, which originates from the proepicardium, gives rise to a set of invasive pluripotent epicardially-derived cells (EPDCs) that contribute to the development of 1) subepicardial mesenchyme, 2) coronary vessels, 3) intermyocardial fibrous tissue, and 4) mesenchyme of the atrioventricular cardiac valve primordia. Here we report that chimeric tracing of proepicardial cells as well as a new experimental procedure reveal a non-proepicardial origin for the epicardial-like cells of the more distal part of the outflow tract of the heart (OFT). This observation is supported by the fact that the expression pattern of mesothelial antigens in the epicardial-like cells of the distal outflow tract is different from the expression pattern in the pro epicardially-derived epicardium. The delay of epicardium and EPDC development using diverse microsurgical procedures leads to abnormal cardiac phenotypes in all cardiac areas colonized by proepicardially-derived epicardium and EPDCs. These results suggest the existence of a signaling role for epicardium and EPDCs in cardiac development and support the hypothesis that ontogenetical diversity of cardiac epicardium is involved in heart chamber morphogenesis and remodeling.

Epithelial‐mesenchymal transitions in the developing heart of the dogfish (Scyliorhinus canicula). A scanning electron microscopic study

An epithelial‐mesenchymal transition is involved in two main morphogenetic events of cardiac morphogenesis, namely the differentiation of the valvuloseptal tissue from the endocardial endothelium, and the formation of subepicardial mesenchyme from the epicardial mesothelium. We have proposed that the dogfish (Scyliorhinus canicula) is a suitable model for the study of basic processes of cardiac morphogenesis in vertebrates, since the heart of this primitive fish probably outlines the original bauplan of the vertebrate heart. In order to study in this model the endocardial and epicardial epithelial‐mesenchymal transition under scanning electron microscopy, we have used a technique of paraffin‐embedding, partial sectioning, dewaxing and critical‐point drying. Our results showed: 1) A centrifugal pattern of epicardial development from the atrioventricular groove to the sinus venosus and conus arteriosus; 2) A close spatial and temporal relationship between the endocardial and epicardial epithelial‐mesenchymal transition, although the transformation of the endocardium starts earlier and ends later the epicardial transformation; 3) A complex arrangement of the fibrous extracellular matrix which is established prior to the migration of the mesenchymal cells. Subepicardial, but not subendothelial mesenchymal cells, coalesce in unicellular or pluricellular ring‐like structures that probably are related to the origin of the cardiac vessels.

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

Morris (J. Anat., 1976;121:47–64) proposed that the subepicardial mesenchyme might represent a continuing source of myocardioblasts during embryonic and fetal development. Recent studies have shown that the epicardium and subepicardial mesenchyme, and the coronary vasculature are all derived from a region of the pericardial wall, called the proepicardial serosa. In avian embryos, the cells from the proepicardial serosa colonize the heart via a secondary tissue bridge formed by attachment of proepicardial villi to the heart. In the present study, Morris's hypothesis was tested by tracing the fate of the proepicardial serosa. This was achieved by constructing quail-chick chimeras. The proepicardial serosa was transplanted from HH16/17 quail embryos to HH16/17 chick embryos (ED3). A new transplantation technique facilitated an orthotopic attachment of the quail proepicardial villi to the chicken heart, and prevented the attachment of the chicken proepicardial villi to the heart. The fate of the grafted quail cells was traced in chimeras from ED4 to ED18 with immunohistochemistry, using quail-specific antibodies (QCPN, QH-1). From ED4 onward, the transplant was connected to the dorsal heart wall via its proepicardial villi. Starting from the point of attachment of the quail proepicardial villi to the heart, the originally naked myocardium became almost completely covered by quail-derived epicardium, and quail mesenchymal cells populated the subepicardial, myocardial, and subendocardial layers including the av-endocardial cushions. Quail cells formed the endothelial and smooth muscles cells of the coronary vessels, and the perivascular and intramyocardial fibroblasts. Quail myocardial cells were never found in the subepicardial, myocardial, and subendocardial layers. This suggests that the subepicardial mesenchyme normally does not contribute a substantial number of myocardioblasts to the developing avian heart. The new transplantation technique presented facilitates the production of chimeric hearts in which the derivatives of the proepicardial serosa are almost completely of donor origin. Thistechnique might be useful for future studies analyzing the role of certain genes in cardiac development by the creation of somatic transgenics. Anat Rec 255:212–226, 1999. © 1999 Wiley-Liss, Inc.

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.

Morris (J. Anat., 1976;121:47-64) proposed that the subepicardial mesenchyme might represent a continuing source of myocardioblasts during embryonic and fetal development. Recent studies have shown that the epicardium and subepicardial mesenchyme, and the coronary vasculature are all derived from a region of the pericardial wall, called the proepicardial serosa. In avian embryos, the cells from the proepicardial serosa colonize the heart via a secondary tissue bridge formed by attachment of proepicardial villi to the heart. In the present study, Morris's hypothesis was tested by tracing the fate of the proepicardial serosa. This was achieved by constructing quail-chick chimeras. The proepicardial serosa was transplanted from HH16/17 quail embryos to HH16/17 chick embryos (ED3). A new transplantation technique facilitated an orthotopic attachment of the quail proepicardial villi to the chicken heart, and prevented the attachment of the chicken proepicardial villi to the heart. The fate of the grafted quail cells was traced in chimeras from ED4 to ED18 with immunohistochemistry, using quail-specific antibodies (QCPN, QH-1). From ED4 onward, the transplant was connected to the dorsal heart wall via its proepicardial villi. Starting from the point of attachment of the quail proepicardial villi to the heart, the originally naked myocardium became almost completely covered by quail-derived epicardium, and quail mesenchymal cells populated the subepicardial, myocardial, and subendocardial layers including the av-endocardial cushions. Quail cells formed the endothelial and smooth muscles cells of the coronary vessels, and the perivascular and intramyocardial fibroblasts. Quail myocardial cells were never found in the subepicardial, myocardial, and subendocardial layers. This suggests that the subepicardial mesenchyme normally does not contribute a substantial number of myocardioblasts to the developing avian heart. The new transplantation technique presented facilitates the production of chimeric hearts in which the derivatives of the proepicardial serosa are almost completely of donor origin. This technique might be useful for future studies analyzing the role of certain genes in cardiac development by the creation of somatic transgenics.

Immunoreactivity of the ets-1 transcription factor correlates with areas of epithelial-mesenchymal transition in the developing avian heart.

Cardiac morphogenesis involves substantial remodeling processes that include cell transdifferentiation and migration. The c-ets-1 protooncogene codes for a transcription factor that can transactivate a number of genes involved in developmental processes such as degradation of extracellular matrices and cell migration. We have immunolocated the ets-1 protein in the heart of quail and chick embryos between the Hamburger and Hamilton stages HH16 and HH37. In HH16-17 embryos, the ets-1 transcription factor was only detected in some endocardial cells and in most mesothelial and mesenchymal cells of the proepicardium. Ets-1 immunoreactivity increased markedly in the developing endocardial cushions, myocardium, epicardium and early subepicardial mesenchyme of HH18-19 embryos. By HH20-24 the immunoreactivity was found throughout the heart, with a stronger intensity in the areas of epithelial-mesenchymal transition of the endocardium and epicardium. In embryos between HH26 and HH33, ets-1 immunoreactivity increased in the cushion mesenchyme, atrioventricular endocardium, ventricular epicardium and subepicardial mesenchyme cells, but not in other areas of the heart. The immunoreactivity declined in the innermost part of the endocardial cushions. The subepicardial mesenchyme was particularly immunoreactive in these stages, coinciding with the development of the subepicardial vascular network. In fact, ets-1 colocalized with the quail vascular marker QH1 in the subepicardial mesenchymal cells. Ets-1-negative cells were abundant in the subepicardium and valvuloseptal tissue of the HH37 embryos. The results suggest that ets-1, probably through transactivation of genes such as urokinase-type plasminogen activator and matrix metalloproteinases, might play a crucial role in the differentiation of the cushion and subepicardial mesenchyme, the formation of the intratrabecular sinusoids and the early development of the cardiac vessels.

The Origin of the Subepicardial Mesenchyme in the Avian Embryo: An Immunohistochemical and Quail–Chick Chimera Study

It has been proposed that the subepicardial mesenchymal cells (SEMC) originate from the primitive epicardium and also from migration of extracardiac mesenchyme from the liver area. We have studied the possibility of an origin of SEMC through transformation of the proepicardial mesothelium, as well as the potential of the early proepicardium to generate epicardium and SEMC in quail–chick chimeras. The study was carried out in quail and chick embryos between HH16 and HH29 stages. Most proepicardial cells, mesothelial as well as mesenchymal, were cytokeratin and vimentin immunoreactive, suggesting a cytoskeletal shift from the epithelial to the mesenchymal type. Furthermore, we immunolocated, in the proepicardial mesothelium, three proteins specifically expressed during the endothelial–mesenchymal transition of the endocardial cushions, namely the JB3/fibrillin-associated antigen, the ES/130 protein and the smooth muscle cell α-actin. Grafts of proepicardial tissue from HH16–17 quail embryos into chick embryos of the same age originated large areas of donor-derived epicardium, including mesothelial, mesenchymal, and vascular cells. The donor-derived primitive epicardium showed segment-specific features, being squamous and adhered to the myocardium on the atrial wall and showing morphological signs of ingression in the atrioventricular groove and outflow tract. These morphological traits together with the distribution of vimentin, the ES/130 protein, and the JB3/fibrillin-associated antigen suggested a localized transformation of some epicardial mesothelial cells into mesenchyme. Most of the donor-derived cells, mesothelial and mesenchymal, showed the vascular marker QH1, which frequently colocalized with cytokeratin. Heterotopic grafts of quail splanchnopleura into the pericardial cavity of chick embryos originated a squamous, epicardial-like, cytokeratin-immunoreactive cell layer on the heart surface, as well as a few QH1+subepicardial and intramyocardial cells. The results suggest that a substantial part of the subepicardial mesenchyme, including the progenitors of the cardiac vessels, originates from the transformation of proepicardial and epicardial mesothelial cells into mesenchyme, and that the epicardial transition could be driven by a segment-specific myocardial signal.

Contribution of the primitive epicardium to the subepicardial mesenchyme in hamster and chick embryos.

A study about the hypothetical contribution of the epicardial cells to the subepicardial mesenchyme was carried out in Syrian hamster embryos of 9-12 days post coitum (dpc) and chick embryos of 3-5 days of incubation. In the epicardium and subepicardium of these embryos we have immunolocated the proteins cytokeratin (CK), vimentin (VIM), fibronectin (FN), and two antigens related to the transformation of endocardial cells into valvuloseptal mesenchyme, ES/130 and JB3. In the hamster embryos, CK+ subepicardial mesenchymal cells (SEMC) were apparently migrating from the primitive epicardium from 9.5 dpc at the atrioventricular (AV) groove and proximal outflow tract (OFT). The morphological signs of delamination extended by 11 dpc to the epicardium of the interventricular groove and the dorsal part of the ventricle. The relative abundance of the CK+ SEMC decreased in embryos of 12 dpc. VIM colocalized with CK in most SEMC, and in some epicardial mesothelial cells, mainly at the areas of delamination. CK immunoreactivity was also found in some early subepicardial capillaries. Similar observations were made in the chick embryos studied. The immunoreactive patterns obtained at the subepicardium with anti-FN, ES/130, and JB3 antibodies were similar to those reported in the areas of endothelial transformation of the endocardial cushions. We suggest that these observations are compatible with an epithelial-mesenchymal transformation involving the epicardial mesothelium and originating at least a part of the SEMC.

Development of the subepicardial mesenchyme and the early cardiac vessels in the dogfish (Scyliorhinus canicula)

A study was designed to assess the development of the subepicardial mesenchyme and the early cardiac vessels in the elasmobranch dogfish (Scyliorhinus canicula). The findings showed that the subepicardial mesenchymal cells originated, at least in part, from the primitive epicardium. This process seemed to be more intense where the subepicardium was the widest, namely, at the atrioventricular and conoventricular grooves as well as at the lateral margins of the ventricle. At these sites, the epicardial cells displayed morphological features usually related to epithelial-mesenchymal transitions, i.e., cell hypertrophy, motility-like basal appendages, cell overlapping, intercellular gaps, and acquisition of a secretory phenotype. The epicardial cells which covered other parts of the heart were flattened and showed smaller nuclei; their basal surface was fibronectin-immunoreactive, unlike that of the hypertrophied epicardial cells of the atrioventricular groove. Fibronectin immunoreactivity also developed in the subepicardial space as the mesenchymal cell population increased. In the dogfish, a subepicardial network of capillaries developed subsequent to the epicardial covering of the heart. Before this network was established, numerous capillary-like structures were present in the subepicardial space. These capillary-like structures appeared as single cells with a large vacuole or as connections of cytoplasmic processes of one or several cells by junctional complexes. The cells that formed the capillary-like structures probably originated from the subepicardial mesenchymal cells. The main ultrastructural difference between the mesenchymal cells and the capillary-like structures was the presence, in the latter, of membrane-bound, electron-dense cytoplasmic inclusions 0.2–1.0 μm in diameter. Morphological evidence suggested that both the subepicardial capillary plexus and the endothelial precursors of the adult cardiac veins resulted from the coalescence of capillary-like structures. © 1996 Wiley-Liss, Inc.

Development of the subepicardial mesenchyme and the early cardiac vessels in the dogfish (Scyliorhinus canicula)

A study was designed to assess the development of the subepicardial mesenchyme and the early cardiac vessels in the elasmobranch dogfish (Scyliorhinus canicula). The findings showed that the subepicardial mesenchymal cells originated, at least in part, from the primitive epicardium. This process seemed to be more intense where the subepicardium was the widest, namely, at the atrioventricular and conoventricular grooves as well as at the lateral margins of the ventricle. At these sites, the epicardial cells displayed morphological features usually related to epithelial-mesenchymal transitions, i.e., cell hypertrophy, motility-like basal appendages, cell overlapping, intercellular gaps, and acquisition of a secretory phenotype. The epicardial cells which covered other parts of the heart were flattened and showed smaller nuclei; their basal surface was fibronectin-immunoreactive, unlike that of the hypertrophied epicardial cells of the atrioventricular groove. Fibronectin immunoreactivity also developed in the subepicardial space as the mesenchymal cell population increased. In the dogfish, a subepicardial network of capillaries developed subsequent to the epicardial covering of the heart. Before this network was established, numerous capillary-like structures were present in the subepicardial space. These capillary-like structures appeared as single cells with a large vacuole or as connections of cytoplasmic processes of one or several cells by junctional complexes. The cells that formed the capillary-like structures probably originated from the subepicardial mesenchymal cells. The main ultrastructural difference between the mesenchymal cells and the capillary-like structures was the presence, in the latter, of membrane-bound, electron-dense cytoplasmic inclusions 0.2–1.0 μm in diameter. Morphological evidence suggested that both the subepicardial capillary plexus and the endothelial precursors of the adult cardiac veins resulted from the coalescence of capillary-like structures. © 1996 Wiley-Liss, Inc.

Experimental study on the formation of the epicardium in chick embryos

In chick embryos, the formation of the epicardium proceeds from the attachment of a secondary sinuventricular mesocardium. This mesocardium is formed by the adhesion of pericardial villi with the dorsal surface of the heart. It was the aim of this study to clarify the role of the pericardial villi in the formation of the epicardium. For this purpose, the contact between the pericardial villi and the heart was prevented by placement of a piece of the shell membrane between them. After re-incubation, the hearts of the experimental embryos could be assigned to one of two different groups: hearts completely lacking a secondary mesocardium (Group A), and hearts without the sinu-ventricular but with a dystopic secondary mesocardium (Group B). In Group A, the formation of the epicardium and subepicardial mesenchyme was found to be severely disturbed. In Group B, the formation of the epicardium proceeded from the point of attachment of a dystopic secondary mesocardium; defective development of the subepicardial mesenchyme was not encountered. These results support the view that the epicardium is derived from the pericardial epithelium.