Proepicardial Organ Research Articles

Lineage tracing using Wnt2b-2A-CreERT2 knock-in mice reveals the contributions of Wnt2b-expressing cells to novel subpopulations of mesothelial/epicardial cell lineages during mouse development.

Mesothelial and epicardial cells give rise to various types of mesenchymal cells via epithelial (mesothelial)-to-mesenchymal transition during development. However, the genes controlling the differentiation and diversification of mesothelial/epicardial cells remain unclear. Here, we examined Wnt2b expression in the embryonic mesothelium and epicardium and performed lineage tracing of Wnt2b-expressing cells by using novel Wnt2b-2A-CreERT2 knock-in and LacZ-reporter mice. Wnt2b was expressed in mesothelial cells covering visceral organs, but the expression was restricted in their subpopulations. Wnt2b-expressing cells labeled at embryonic day (E) 10.5 were distributed to the mesothelium and mesenchyme in the lungs, abdominal wall, stomach, and spleen in Wnt2b2A-CreERT2/+;R26RLacZ/+ mice at E13.0. Wnt2b was initially expressed in the proepicardial organ (PEO) at E9.5 and then in the epicardium after E10.0. Wnt2b-expressing PEO cells labeled at E9.5 differentiated into a small fraction of cardiac fibroblasts and preferentially localized at the left side of the postnatal heart. LacZ+ epicardium-derived cells labeled at E10.5 differentiated into a small fraction of fibroblasts and smooth muscle cells in the postnatal heart. Taken together, our results reveal novel subpopulations of PEO and mesothelial/epicardial cells that are distinguishable by Wnt2b expression and elucidate the unique contribution of Wnt2b-expressing PEO and epicardial cells to the postnatal heart.

A patterned human primitive heart organoid model generated by pluripotent stem cell self-organization

Pluripotent stem cell-derived organoids can recapitulate significant features of organ development in vitro. We hypothesized that creating human heart organoids by mimicking aspects of in utero gestation (e.g., addition of metabolic and hormonal factors) would lead to higher physiological and anatomical relevance. We find that heart organoids produced using this self-organization-driven developmental induction strategy are remarkably similar transcriptionally and morphologically to age-matched human embryonic hearts. We also show that they recapitulate several aspects of cardiac development, including large atrial and ventricular chambers, proepicardial organ formation, and retinoic acid-mediated anterior-posterior patterning, mimicking the developmental processes found in the post-heart tube stage primitive heart. Moreover, we provide proof-of-concept demonstration of the value of this system for disease modeling by exploring the effects of ondansetron, a drug administered to pregnant women and associated with congenital heart defects. These findings constitute a significant technical advance for synthetic heart development and provide a powerful tool for cardiac disease modeling.

Abstract 13459: Human iPS-Derived Pre-Epicardial Cells Direct Cardiomyocyte Aggregation Expansion and Organization in vitro

During heart maturation, epicardial-derived cells emanating from the pro-epicardial organ envelope the developing heart, then integrate within the myocardium and undergo epithelial-mesenchymal transition (EMT) to become fibroblasts, smooth muscle cells, and endothelial cells that enable several processes of healthy heart morphogenesis (i.e., ventricular thickening, compaction, and angiogenesis). Directed cardiac differentiation of human induced-pluripotent stem cells (hiPSCs) holds great potential for generating several cardiovascular cell types, which can theoretically be combined to model cardiac development more completely and engineer sophisticated myocardial grafts. Here, we show that differentiating hiPSC-derived lateral plate mesoderm (LPM) with BMP4, RA and VEGF (BVR) for 96 hours can generate a premature form of epicardial cells (termed pre-epicardial cells, PECs) with high efficiency (86.8 ± 4.1% WT1 + by FACS), that also show significant increases in WT1, TBX18, SEMA3D , TBX5, BNC, and SCX transcript expression within 7 days. This protocol was successfully reproduced in three commercial lines of hiPSCs. BVR stimulation after Wnt inhibition of LPM demonstrated efficient co-differentiation and spatial organization of PECs and cardiomyocytes (CMs) in a single 2D culture. Co-culture consolidated CMs into dense aggregates, which then formed a connected beating syncytium with significantly enhanced contractility (0.029 ± 0.002 mN/mm 2 ; p<0.05 vs. CMs-alone and co-culture controls), improved calcium handling (i.e. amplitude, upstroke, and decay), sarcomere length (1.82 ± 0.02 μm vs. 1.72 ± 0.03 μm, p=0.00264) and mitochondrial density (79.4 ± 3% vs. 54.9 ± 6%, p=0.0029). Co-cultured PECs demonstrated enhanced maturity to epicardial cells with significant upregulation of UPK1B, ITGA4, and ALDH1A2 transcript expressions. Our study also demonstrated that PECs secrete IGF2 and stimulate CM proliferation in co-culture (37.2 ± 2.1% vs. 28.1 ± 1%, p=0.016). These characteristics suggest PECs could play a key role in enhancing cell-cell signaling and tissue organization, which may help to recapitulate important aspects of cardiac morphogenesis in vitro and advance the maturation of bioengineered cardiac constructs.

Generating Self-Assembling Human Heart Organoids Derived from Pluripotent Stem Cells.

The ability to study human cardiac development in health and disease is highly limited by the capacity to model the complexity of the human heart in vitro. Developing more efficient organ-like platforms that can model complex in vivo phenotypes, such as organoids and organs-on-a-chip, will enhance the ability to study human heart development and disease. This paper describes a protocol to generate highly complex human heart organoids (hHOs) by self-organization using human pluripotent stem cells and stepwise developmental pathway activation using small molecule inhibitors. Embryoid bodies (EBs) are generated in a 96-well plate with round-bottom, ultra-low attachment wells, facilitating suspension culture of individualized constructs. The EBs undergo differentiation into hHOs by a three-step Wnt signaling modulation strategy, which involves an initial Wnt pathway activation to induce cardiac mesoderm fate, a second step of Wnt inhibition to create definitive cardiac lineages, and a third Wnt activation step to induce proepicardial organ tissues. These steps, carried out in a 96-well format, are highly efficient, reproducible, and produce large amounts of organoids per run. Analysis by immunofluorescence imaging from day 3 to day 11 of differentiation reveals first and second heart field specifications and highly complex tissues inside hHOs at day 15, including myocardial tissue with regions of atrial and ventricular cardiomyocytes, as well as internal chambers lined with endocardial tissue. The organoids also exhibit an intricate vascular network throughout the structure and an external lining of epicardial tissue. From a functional standpoint, hHOs beat robustly and present normal calcium activity as determined by Fluo-4 live imaging. Overall, this protocol constitutes a solid platform for in vitro studies in human organ-like cardiac tissues.

Genetic and Cellular Interaction During Cardiovascular Development Implicated in Congenital Heart Diseases.

Congenital heart disease (CHD) is the most common life-threatening congenital anomaly. CHD occurs due to defects in cardiovascular development, and the majority of CHDs are caused by a multifactorial inheritance mechanism, which refers to the interaction between genetic and environmental factors. During embryogenesis, the cardiovascular system is derived from at least four distinct cell lineages: the first heart field, second heart field, cardiac neural crest, and proepicardial organ. Understanding the genes involved in each lineage is essential to uncover the genomic architecture of CHD. Therefore, we provide an overview of recent research progress using animal models and mutation analyses to better understand the molecular mechanisms and pathways linking cardiovascular development and CHD. For example, we highlight our recent work on genes encoding three isoforms of inositol 1,4,5-trisphosphate receptors (IP3R1, 2, and 3) that regulate various vital and developmental processes, which have genetic redundancy during cardiovascular development. Specifically, IP3R1 and 2 have redundant roles in the atrioventricular cushion derived from the first heart field lineage, whereas IP3R1 and 3 exhibit redundancy in the right ventricle and the outflow tract derived from the second heart field lineage, respectively. Moreover, 22q11.2 deletion syndrome (22q11DS) is highly associated with CHD involving the outflow tract, characterized by defects of the cardiac neural crest lineage. However, our studies have shown that TBX1, a major genetic determinant of 22q11DS, was not expressed in the cardiac neural crest but rather in the second heart field, suggesting the importance of the cellular interaction between the cardiac neural crest and the second heart field. Comprehensive genetic analysis using the Japanese genome bank of CHD and mouse models revealed that a molecular regulatory network involving GATA6, FOXC1/2, TBX1, SEMA3C, and FGF8 was essential for reciprocal signaling between the cardiac neural crest and the second heart field during cardiovascular development. Elucidation of the genomic architecture of CHD using induced pluripotent stem cells and next-generation sequencing technology, in addition to genetically modified animal models and human mutation analyses, would facilitate the development of regenerative medicine and/or preventive medicine for CHD in the near future.

Spatiotemporal Analysis Reveals Overlap of Key Proepicardial Markers in the Developing Murine Heart.

SummaryThe embryonic epicardium, originating from the proepicardial organ (PEO), provides a source of multipotent progenitors for cardiac lineages, including pericytes, fibroblasts, and vascular smooth muscle cells. Maximizing the regenerative capacity of the adult epicardium depends on recapitulating embryonic cell fates. The potential of the epicardium to contribute coronary endothelium is unclear, due to conflicting Cre-based lineage trace data. Controversy also surrounds when epicardial cell fate becomes restricted. Here, we systematically investigate expression of five widely used epicardial markers, Wt1, Tcf21, Tbx18, Sema3d, and Scx, over the course of development. We show overlap of markers in all PEO and epicardial cells until E13.5, and find no evidence for discrete proepicardial sub-compartments that might contribute coronary endothelium via the epicardial layer. Our findings clarify a number of prevailing discrepancies and support the notion that epicardium-derived cell fate, to form fibroblasts or mural cells, is specified after epithelial-mesenchymal transition, not pre-determined within the PEO.

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.

Epicardium in Heart Development.

The epicardium, the outermost tissue layer that envelops all vertebrate hearts, plays a crucial role in cardiac development and regeneration and has been implicated in potential strategies for cardiac repair. The heterogenous cell population that composes the epicardium originates primarily from a transient embryonic cell cluster known as the proepicardial organ (PE). Characterized by its high cellular plasticity, the epicardium contributes to both heart development and regeneration in two critical ways: as a source of progenitor cells and as a critical signaling hub. Despite this knowledge, there are many unanswered questions in the field of epicardial biology, the resolution of which will advance the understanding of cardiac development and repair. We review current knowledge in cross-species epicardial involvement, specifically in relation to lineage specification and differentiation during cardiac development.

Scleraxis regulates Twist1 and Snai1 expression in the epithelial-to-mesenchymal transition.

Numerous physiological and pathological events, from organ development to cancer and fibrosis, are characterized by an epithelial-to-mesenchymal transition (EMT), whereby adherent epithelial cells convert to migratory mesenchymal cells. During cardiac development, proepicardial organ epithelial cells undergo EMT to generate fibroblasts. Subsequent stress or damage induces further phenotype conversion of fibroblasts to myofibroblasts, causing fibrosis via synthesis of an excessive extracellular matrix. We have previously shown that the transcription factor scleraxis is both sufficient and necessary for the conversion of cardiac fibroblasts to myofibroblasts and found that scleraxis knockout reduced cardiac fibroblast numbers by 50%, possibly via EMT attenuation. Scleraxis induced expression of the EMT transcriptional regulators Twist1 and Snai1 via an unknown mechanism. Here, we report that scleraxis binds to E-box consensus sequences within the Twist1 and Snai1 promoters to transactivate these genes directly. Scleraxis upregulates expression of both genes in A549 epithelial cells and in cardiac myofibroblasts. Transforming growth factor-β induces EMT, fibrosis, and scleraxis expression, and we found that transforming growth factor-β-mediated upregulation of Twist1 and Snai1 completely depends on the presence of scleraxis. Snai1 knockdown upregulated the epithelial marker E-cadherin; however, this effect was lost after scleraxis overexpression, suggesting that scleraxis may repress E-cadherin expression. Together, these results indicate that scleraxis can regulate EMT via direct transactivation of the Twist1 and Snai1 genes. Given the role of scleraxis in also driving the myofibroblast phenotype, scleraxis appears to be a critical controller of fibroblast genesis and fate in the myocardium and thus may play key roles in wound healing and fibrosis. NEW & NOTEWORTHY The molecular mechanism by which the transcription factor scleraxis mediates Twist1 and Snai1 gene expression was determined. These results reveal a novel means of transcriptional regulation of epithelial-to-mesenchymal transition and demonstrate that transforming growth factor-β-mediated epithelial-to-mesenchymal transition is dependent on scleraxis, providing a potential target for controlling this process.

Avian coronary endothelium is a mosaic of sinus venosus- and ventricle-derived endothelial cells in a region-specific manner.

The origin of coronary endothelial cells (ECs) has been investigated in avian species, and the results showed that the coronary ECs originate from the proepicardial organ (PEO) and developing epicardium. Genetic approaches in mouse models showed that the major source of coronary ECs is the sinus venosus endothelium or ventricular endocardium. To clarify and reconcile the differences between avian and mouse species, we examined the source of coronary ECs in avian embryonic hearts. Using an enhanced green fluorescent protein-Tol2 system and fluorescent dye labeling, four types of quail-chick chimeras were made and quail-specific endothelial marker (QH1) immunohistochemistry was performed. The developing PEO consisted of at least two cellular populations in origin, one was sinus venosus endothelium-derived inner cells and the other was surface mesothelium-derived cells. The majority of ECs in the coronary stems, ventricular free wall, and dorsal ventricular septum originated from the sinus venosus endothelium. The ventricular endocardium contributed mainly to the septal artery and a few cells to the coronary stems. Surface mesothelial cells of the PEO differentiated mainly into a smooth muscle phenotype, but a few differentiated into ECs. In avian species, the coronary endothelium had a heterogeneous origin in a region-specific manner, and the sources of ECs were basically the same as those observed in mice.

β-Catenin stabilization promotes proliferation and increase in cardiomyocyte number in chick embryonic epicardial explant culture.

Cardiomyocyte (CM) differentiation from proepicardial organ- (PEO) and embryonic epicardium (eEpi)-derived cells or EPDCs in a developing heart emerges as a wide interest in purview of cardiac repair and regenerative medicine. eEpi originates from the precursor PEO and EPDCs, which contribute to several cardiac cell types including smooth muscle cells, fibroblasts, endothelial cells, and CMs during cardiogenesis. Here in this report, we have analyzed several cardiac lineage-specific marker gene expressions between PEO and eEpi cells. We have found that PEO-derived cells show increased level of CM lineage-specific marker gene expression compared to eEpi cells. Moreover, Wnt signaling activation results in increased level of CM-specific marker gene expression in both PEO and eEpi cells in culture. Interestingly, Wnt signaling activation also increases the number of proliferating and sarcomeric myosin (Mf20)-positive cells in eEpi explant culture. Together, this data suggests that eEpi cells as a source for CM differentiation and Wnt signaling mediator, β-catenin, might play an important role in CM differentiation from eEpi cells in culture.

Wtip is required for proepicardial organ specification and cardiac left/right asymmetry in zebrafish.

Wilm's tumor 1 interacting protein (Wtip) was identified as an interacting partner of Wilm's tumor protein (WT1) in a yeast two-hybrid screen. WT1 is expressed in the proepicardial organ (PE) of the heart, and mouse and zebrafish wt1 knockout models appear to lack the PE. Wtip's role in the heart remains unexplored. In the present study, we demonstrate that wtip expression is identical in wt1a-, tcf21-, and tbx18-positive PE cells, and that Wtip protein localizes to the basal body of PE cells. We present the first genetic evidence that Wtip signaling in conjunction with WT1 is essential for PE specification in the zebrafish heart. By overexpressing wtip mRNA, we observed ectopic expression of PE markers in the cardiac and pharyngeal arch regions. Furthermore, wtip knockdown embryos showed perturbed cardiac looping and lacked the atrioventricular (AV) boundary. However, the chamber-specific markers amhc and vmhc were unaffected. Interestingly, knockdown of wtip disrupts early left-right (LR) asymmetry. Our studies uncover new roles for Wtip regulating PE cell specification and early LR asymmetry, and suggest that the PE may exert non-autonomous effects on heart looping and AV morphogenesis. The presence of cilia in the PE, and localization of Wtip in the basal body of ciliated cells, raises the possibility of cilia-mediated PE signaling in the embryonic heart.

A glimpse of Cre-mediated controversies in epicardial signalling.

Among the many important aspects of cardiac development, formation of the epicardium and the underlining signalling pathways that direct the mobilization and differentiation of epicardial cells have been one of the highly pursued research subjects within the past decade. These epicardium-derived cells (EPDCs) are reported to contribute to several critical cell types which include cardiac myofibroblasts and coronary smooth muscle contributing to the formation of the coronary vasculature network within the developing embryonic heart.1 Recent data suggesting that epicardial progenitor cells (EPCs) can contribute to newly formed cardiomyocytes in injured hearts2,3 has drawn further attention into the mechanisms of epicardiogenesis, as the successful use of epicardial cells in cell replacement therapies will undoubtedly have a high clinical impact on treating heart disease. To achieve this goal, a better understanding of the molecular mechanism that drive epicardiogenesis will be required. As the outermost cell layer of the vertebrate heart, the epicardium is derived from the proepicardial organ (PE), which originates from splanchnic mesenchyme within the septum transversum that is juxtaposed near the venous pole of the embryonic day 9.5 (E9.5) mouse heart (E22 for human). EPCs detach from the PE, migrate into pericardial cavity, and adhere to the myocardial surface where they rapidly spread to form a mesothelial monolayer covering the atria, atrioventricular canal, and ventricles, and in doing so, establish a primary epicardium by E10.5 in the mouse. Two days later, at E12.5, some of the primary epicardial cells undergo an epithelial to mesenchymal transition to convert into highly mobile and developmentally plastic EPDCs ( Figure 1 ). These EPDCs proceed to invade the subepicardial matrix and subsequently migrate into the myocardium, where they differentiate into interstitial …

The Lhx9-integrin pathway is essential for positioning of the proepicardial organ.

The development of the vertebrate embryonic heart occurs by hyperplastic growth as well as the incorporation of cells from tissues outside of the initial heart field. Amongst these tissues is the epicardium, a cell structure that develops from the precursor proepicardial organ on the right side of the septum transversum caudal to the developing heart. During embryogenesis, cells of the proepicardial organ migrate, adhere and envelop the maturing heart, forming the epicardium. The cells of the epicardium then delaminate and incorporate into the heart giving rise to cardiac derivatives, including smooth muscle cells and cardiac fibroblasts. Here, we demonstrate that the LIM homeodomain protein Lhx9 is transiently expressed in Xenopus proepicardial cells and is essential for the position of the proepicardial organ on the septum transversum. Utilizing a small-molecule screen, we found that Lhx9 acts upstream of integrin-paxillin signaling and consistently demonstrate that either loss of Lhx9 or disruption of the integrin-paxillin pathway results in mis-positioning of the proepicardial organ and aberrant deposition of extracellular matrix proteins. This leads to a failure of proepicardial cell migration and adhesion to the heart, and eventual death of the embryo. Collectively, these studies establish a requirement for the Lhx9-integrin-paxillin pathway in proepicardial organ positioning and epicardial formation.

Hopx and the Cardiomyocyte Parentage

The dissection of steps that pattern primitive mesoderm toward the cardiovascular lineages is important to our current understanding of cardiovascular development and can instruct regenerative approaches to heart repair.1 Primitive precursors that can adopt a cardiovascular fate, but also a subset of other mesoderm, are marked by the transcription factor Mesp1 (refs. 2, 3). Later precursors, which selectively give rise to two or more of the cardiovascular cell types (bi- and multipotent cardiovascular progenitor cells), are marked by the transcription factors Nkx2.5 or Isl1, indicators of the first and second heart-forming regions of the embryo (“heart fields”).4,5,6 What has been missing, however, is a molecular feature that uniquely marks cells destined to become cardiomyocytes within the heart. To illustrate, mapping the descendants of cells expressing either Nkx2.5 or Isl1, using the Cre/lox system to indelibly label their progeny in vivo, marks virtually all cell types of the mature heart, including smooth muscle, endothelium, epicardium, and fibroblasts.1 This ubiquity results from expression of Nkx2.5 and Isl1 in the proepicardial organ,7 a specialized structure on the surface of the early heart that gives rise to the external lining of the heart (epicardium) and its derivatives (coronary smooth muscle, coronary endothelial cells, cardiac fibroblasts). In short, what is missing is a marker of the immediate and specific precursor of cardiomyocytes, rather than earlier multipotent cells. A recent report by Jain et al. now shows that an atypical homeodomain transcription factor, Hopx, lacking a motif for direct DNA binding, is restricted to the direct precursors of cardiomyocytes, and not to those of the various other cardiac cells.8

Numb family proteins: novel players in cardiac morphogenesis and cardiac progenitor cell differentiation

Vertebrate heart formation is a spatiotemporally regulated morphogenic process that initiates with bilaterally symmetric cardiac primordial cells migrating toward the midline to form a linear heart tube. The heart tube then elongates and undergoes a series of looping morphogenesis, followed by expansions of regions that are destined to become primitive heart chambers. During the cardiac morphogenesis, cells derived from the first heart field contribute to the primary heart tube, and cells from the secondary heart field, cardiac neural crest, and pro-epicardial organ are added to the heart tube in a precise spatiotemporal manner. The coordinated addition of these cells and the accompanying endocardial cushion morphogenesis yield the atrial, ventricular, and valvular septa, resulting in the formation of a four-chambered heart. Perturbation of progenitor cells' deployment and differentiation leads to a spectrum of congenital heart diseases. Two of the genes that were recently discovered to be involved in cardiac morphogenesis are Numb and Numblike. Numb, an intracellular adaptor protein, distinguishes sibling cell fates by its asymmetric distribution between the two daughter cells and its ability to inhibit Notch signaling. Numb regulates cardiac progenitor cell differentiation in Drosophila and controls heart tube laterality in Zebrafish. In mice, Numb and Numblike, the Numb family proteins (NFPs), function redundantly and have been shown to be essential for epicardial development, cardiac progenitor cell differentiation, outflow tract alignment, atrioventricular septum morphogenesis, myocardial trabeculation, and compaction. In this review, we will summarize the functions of NFPs in cardiac development and discuss potential mechanisms of NFPs in the regulation of cardiac development.

Tbx5 Is Required for Avian and Mammalian Epicardial Formation and Coronary Vasculogenesis

Holt-Oram syndrome is an autosomal dominant heart-hand syndrome caused by mutations in the TBX5 gene. Overexpression of Tbx5 in the chick proepicardial organ impaired coronary blood vessel formation. However, the potential activity of Tbx5 in the epicardium itself, and the role of Tbx5 in mammalian coronary vasculogenesis, remains largely unknown. To evaluate the consequences of altered Tbx5 gene dosage during proepicardial organ and epicardial development in the embryonic chick and mouse. Retroviral-mediated knockdown or upregulation of Tbx5 expression in the embryonic chick proepicardial organ and proepicardial-specific deletion of Tbx5 in the embryonic mouse (Tbx5(epi-/)) impaired normal proepicardial organ cell development, inhibited epicardial and coronary blood vessel formation, and altered developmental gene expression. The generation of epicardial-derived cells and their migration into the myocardium were impaired between embryonic day (E) 13.5 to 15.5 in mutant hearts because of delayed epicardial attachment to the myocardium and subepicardial accumulation of epicardial-derived cells. This caused defective coronary vasculogenesis associated with impaired vascular smooth muscle cell recruitment and reduced invasion of cardiac fibroblasts and endothelial cells into myocardium. In contrast to wild-type hearts that exhibited an elaborate ventricular vascular network, Tbx5(epi-/-) hearts displayed a marked decrease in vascular density that was associated with myocardial hypoxia as exemplified by hypoxia inducible factor-1α upregulation and increased binding of hypoxyprobe-1. Tbx5(epi-/-) mice with such myocardial hypoxia exhibited reduced exercise capacity when compared with wild-type mice. Our findings support a conserved Tbx5 dose-dependent requirement for both proepicardial and epicardial progenitor cell development in chick and in mouse coronary vascular formation.

Evolution and development of ventricular septation in the amniote heart.

During cardiogenesis the epicardium, covering the surface of the myocardial tube, has been ascribed several functions essential for normal heart development of vertebrates from lampreys to mammals. We investigated a novel function of the epicardium in ventricular development in species with partial and complete septation. These species include reptiles, birds and mammals. Adult turtles, lizards and snakes have a complex ventricle with three cava, partially separated by the horizontal and vertical septa. The crocodilians, birds and mammals with origins some 100 million years apart, however, have a left and right ventricle that are completely separated, being a clear example of convergent evolution. In specific embryonic stages these species show similarities in development, prompting us to investigate the mechanisms underlying epicardial involvement. The primitive ventricle of early embryos becomes septated by folding and fusion of the anterior ventricular wall, trapping epicardium in its core. This folding septum develops as the horizontal septum in reptiles and the anterior part of the interventricular septum in the other taxa. The mechanism of folding is confirmed using DiI tattoos of the ventricular surface. Trapping of epicardium-derived cells is studied by transplanting embryonic quail pro-epicardial organ into chicken hosts. The effect of decreased epicardium involvement is studied in knock-out mice, and pro-epicardium ablated chicken, resulting in diminished and even absent septum formation. Proper folding followed by diminished ventricular fusion may explain the deep interventricular cleft observed in elephants. The vertical septum, although indistinct in most reptiles except in crocodilians and pythonidsis apparently homologous to the inlet septum. Eventually the various septal components merge to form the completely septated heart. In our attempt to discover homologies between the various septum components we aim to elucidate the evolution and development of this part of the vertebrate heart as well as understand the etiology of septal defects in human congenital heart malformations.

Epicardium is required for sarcomeric maturation and cardiomyocyte growth in the ventricular compact layer mediated by transforming growth factor β and fibroblast growth factor before the onset of coronary circulation

The epicardium, which is derived from the proepicardial organ (PE) as the third epithelial layer of the developing heart, is crucial for ventricular morphogenesis. An epicardial deficiency leads to a thin compact layer for the developing ventricle; however, the mechanisms leading to the impaired development of the compact layer are not well understood. Using chick embryonic hearts, we produced epicardium-deficient hearts by surgical ablation or blockade of the migration of PE and examined the mechanisms underlying a thin compact myocardium. Sarcomeric maturation (distance between Z-lines) and cardiomyocyte growth (size) were affected in the thin compact myocardium of epicardium-deficient ventricles, in which the amounts of phospho-smad2 and phospho-ERK as well as expression of transforming growth factor (TGF)β2 and fibroblast growth factor (FGF)2 were reduced. TGFβ and FGF were required for the maturation of sarcomeres and growth of cardiomyocytes in cultured ventricles. In ovo co-transfection of dominant negative (dN)-Alk5 (dN-TGFβ receptor I) and dN-FGF receptor 1 to ventricles caused a thin compact myocardium. Our results suggest that immature sarcomeres and small cardiomyocytes are the causative architectures of an epicardium-deficient thin compact layer and also that epicardium-dependent signaling mediated by TGFβ and FGF plays a role in the development of the ventricular compact layer before the onset of coronary circulation.

Dynamic haematopoietic cell contribution to the developing and adult epicardium.

The epicardium is a cellular source with the potential to reconstitute lost cardiovascular tissue following myocardial infarction. Here we show that the adult epicardium contains a population of CD45+ haematopoietic cells (HCs), which are located proximal to coronary vessels and encased by extracellular matrix (ECM). This complex tertiary structure is established during the regenerative window between post-natal days 1 and 7. We show that these HCs proliferate within the first 24 h and are released between days 2 and 7 after myocardial infarction. The ECM subsequently reforms to encapsulate HCs after 21 days. Vav1-tdTomato labelling reveals an integral contribution of CD45+ HCs to the developing epicardium, which is not derived from the proepicardial organ. Transplantation experiments with either whole bone marrow or a Vav1+ subpopulation of cells confirm a contribution of HCs to the intact adult epicardium, which is elevated during the first 24 weeks of adult life but depleted in aged mice.