Immunohistochemical Study of the Origin of the Subepicardial Mesenchyme in the Dogfish (Scyliorhinus canicula)

A mesenchyme appears at the subepicardium of the vertebrate embryo closely following the epicardial ensheathing of the heart. We have suggested earlier that a localized epithelial-mesenchymal transition (= transdifferentialion) of the epicardial mesothelium might explain the origin of the subepicardial mesenchymal cells (SEMC) in a primitive vertebrate, the dogfish Scyliorhinus canicula. We have tested our hypothesis through the immunolocalization, in the embryonic dogfish heart, of three proteins involved in the epithelial—mesenchymal transitions. Fibronectin immunoreactivity (FN-IR) was present between epicardium and myocardium from the earliest stages of the epicardial development. However, in embryos ranging between 20 and 35 mm TL, FN-IR disappeared from the basal surface of the epicardial cells at the atrioventricular and conoventricular grooves and increased in association with the SEMC. Proliferating-cell nuclear antigen immunoreactivity was intense in most epicardial cells and SEMC in these areas and developmental stages, but it declined in later embryos. The JB3 antigen, a fibrillin-related protein, was delected in the cytoplasm and basal surface of the epicardial cells, as well as on the SEMC surface. These immunohistochemical patterns were remarkably similar at the subepicardium and at the endocardial cushions, which are populated by mesenchymal cells derived from the transdifferentiation of the endocardium. © 1998 The Royal Swedish Academy of Sciences. Published by Elsevier Science Ltd. All rights reserved     

Dynamic changes in endoglin expression in the developing mouse heart

Endoglin (ENG) is essential for cardiovascular development and is expressed in the heart from its earliest developmental stages. ENG expression has been reported in the cardiac crescent, endocardium, valve mesenchyme and coronary vascular endothelial cells. However, its expression in these cell types is non-uniform and the dynamic changes in ENG expression during heart development have not been systematically studied.Using immunofluorescent staining we tracked ENG protein expression in mouse embryonic hearts aged from 11.5 to 17.5 days, and in postnatal and adult hearts. ENG is expressed in the endocardium and in venous endothelial cells throughout these developmental stages. ENG protein is down-regulated by approximately two-fold as a subset of early coronary veins reprogram to form arteries within the developing myocardium from E13.5. This two-fold higher ratio of ENG protein in veins versus arteries is maintained throughout cardiac development and in the adult heart.ENG is also down-regulated two-fold following mesenchymal transition of endocardial cells to form cardiac valve mesenchyme, whilst expression of the pan-endothelial marker CD31 is completely lost. A subset of epicardial cells (which do not express ENG protein) delaminate and undergo a similar mesenchymal transition to form epicardially derived cells (EPDCs). This transient intra-myocardial mesenchymal cell population expresses low levels of ENG protein, similar to valve mesenchyme.In conclusion, ENG shows dynamic changes of expression in vascular endothelial cells, endocardial cells and mesenchymal cells in the developing heart that vary according to cardiovascular cell type.     

A critical role for the EphA3 receptor tyrosine kinase in heart development

Eph proteins are receptor tyrosine kinases that control changes in cell shape and migration during development. We now describe a critical role for EphA3 receptor signaling in heart development as revealed by the phenotype of EphA3 null mice. During heart development mesenchymal outgrowths, the atrioventricular endocardial cushions, form in the atrioventricular canal. This morphogenetic event requires endocardial cushion cells to undergo an epithelial to mesenchymal transformation (EMT), and results in the formation of the atrioventricular valves and membranous portions of the atrial and ventricular septa. We show that EphA3 knockouts have significant defects in the development of their atrial septa and atrioventricular endocardial cushions, and that these cardiac abnormalities lead to the death of approximately 75% of homozygous EphA3(-/-) mutants. We demonstrate that EphA3 and its ligand, ephrin-A1, are expressed in adjacent cells in the developing endocardial cushions. We further demonstrate that EphA3(-/-) atrioventricular endocardial cushions are hypoplastic compared to wildtype and that EphA3(-/-) endocardial cushion explants give rise to fewer migrating mesenchymal cells than wildtype explants. Thus our results indicate that EphA3 plays a crucial role in the development and morphogenesis of the cells that give rise to the atrioventricular valves and septa.     

A 3-D model of coronary vessel development

Coronary vascular disease is one of the leading causes of mortality and morbidity in the United States. Therefore, a mechanistic understanding of coronary vessel morphogenesis would aid in the innovation of new therapies targeting vascular disorders. Moreover, a functionally equivalent in vitro model system allows for the delineation of the molecular mechanisms that regulate coronary vessel development. In this study, we present a novel in vitro model system. This three-dimensional (3-D) model system consists of a tubular scaffold, which is engineered from type-I collagen and has been optimized to support the growth of embryonic cardiac tissues. In this report, proepicardial (PE) cells, the developmental precursors of coronary vessels, have been isolated from several model species and cultured on this scaffold. In this model system, the PE cells were able to recapitulate several aspects of coronary vessel morphogenesis including epicardial formation, the epicardial to mesenchymal transformation, and de novo coronary vessel development or vasculogenesis. The differentiation of PE cells was characterized using a variety of specific protein markers. The potential uses of this novel coronary developmental model are discussed.     

Positive and negative regulation of epicardial-mesenchymal transformation during avian heart development

In the developing heart, the epicardium is essential for coronary vasculogenesis as it provides precursor cells that become coronary vascular smooth muscle and perivascular fibroblasts. These precursor cells are derived from the epicardium via epithelial-mesenchymal transformation (EMT). The factors that regulate epicardial EMT are unknown. Using a quantitative in vitro collagen gel assay, we show that serum, FGF-1, -2, and -7, VEGF, and EGF stimulate epicardial EMT. TGFbeta-1 stimulates EMT only weakly, while TGFbeta-2 and -3 do not stimulate EMT. TGFbeta-1, -2, or -3 strongly inhibits transformation of epicardial cells stimulated with FGF-2 or heart-conditioned medium. TGFbeta-3 does not block expression of vimentin, a mesenchymal marker, but appears to inhibit EMT by blocking epithelial cell dissociation and subsequent extracellular matrix invasion. Blocking antisera directed against FGF-1, -2, or -7 substantially inhibit conditioned medium-stimulated EMT in vitro, while antibodies to TGFbeta-1, -2, or -3 increase it. We confirmed FGF stimulation and TGFbeta inhibition of epicardial EMT in organ culture. Immunoblot analysis confirmed the presence of FGF-1, -2, and -7 and TGFbeta-1, -2, and -3 in conditioned medium, and we localized these growth factors to the myocardium and epicardium of stage-appropriate embryos by immunofluorescence. Our results strongly support a model in which myocardially derived FGF-1, -2, or -7 promotes epicardial EMT, while TGFbeta-1, -2, or -3 restrains it. Epicardial EMT appears to be regulated through a different signaling pathway than endocardial EMT.     

Bmp6 and Bmp7 are required for cushion formation and septation in the developing mouse heart.

The mature heart valves and septa are derived from the cardiac cushions which initially form as local outgrowths of mesenchymal cells within the outflow tract and atrioventricular regions. Endocardial cells respond to signals from the overlying myocardium and undergo an epithelial-to-mesenchymal transformation to invade the intervening extracellular matrix. The molecules that can induce and maintain these cell populations are not known, but many candidates, including several TGFbetas and BMPs, have been proposed based on their expression patterns and activities in other systems. In the present study, we describe the expression of Bmp6 and Bmp7 in overlapping and adjacent sites, including the cardiac cushions during mouse embryonic development. Previous analyses demonstrate that neither of these BMPs is required during cardiogenesis, but analysis of Bmp6;Bmp7 double mutants uncovers a marked delay in the formation of the outflow tract endocardial cushions. A proportion of Bmp6;Bmp7 mutants also display defects in valve morphogenesis and chamber septation, and the embryos die between 10.5 and 15.5 dpc due to cardiac insufficiency. These data provide the first genetic evidence that BMPs are involved in the formation of the cardiac cushions.     

Elevated glucose inhibits VEGF-A-mediated endocardial cushion formation: modulation by PECAM-1 and MMP-2

Atrioventricular (AV) septal defects resulting from aberrant endocardial cushion (EC) formation are observed at increased rates in infants of diabetic mothers. EC formation occurs via an epithelial-mesenchymal transformation (EMT), involving transformation of endocardial cells into mesenchymal cells, migration, and invasion into extracellular matrix. Here, we report that elevated glucose inhibits EMT by reducing myocardial vascular endothelial growth factor A (VEGF-A). This effect is reversed with exogenous recombinant mouse VEGF-A165, whereas addition of soluble VEGF receptor-1 blocks EMT. We show that disruption of EMT is associated with persistence of platelet endothelial cell adhesion molecule-1 (PECAM-1) and decreased matrix metalloproteinase-2 (MMP-2) expression. These findings correlate with retention of a nontransformed endocardial sheet and lack of invasion. The MMP inhibitor GM6001 blocks invasion, whereas explants from PECAM-1 deficient mice exhibit MMP-2 induction and normal EMT in high glucose. PECAM-1-negative endothelial cells are highly motile and express more MMP-2 than do PECAM-1-positive endothelial cells. During EMT, loss of PECAM-1 similarly promotes single cell motility and MMP-2 expression. Our findings suggest that high glucose-induced inhibition of AV cushion morphogenesis results from decreased myocardial VEGF-A expression and is, in part, mediated by persistent endocardial cell PECAM-1 expression and failure to up-regulate MMP-2 expression.     

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.     

Degradation of type IV collagen by matrix metalloproteinases is an important step in the epithelial-mesenchymal transformation of the endocardial cushions

Morphogenesis of some tissues and organs in the developing embryo requires the transformation of epithelial cells into mesenchyme followed by cell motility and invasion of surrounding connective tissues. Details of the mechanisms involved in this important process are beginning to be elucidated. The epithelial-mesenchymal transformation (EMT) process involves many steps, one of which is the upregulation and activation of specific extracellular proteinases including members of the matrix metalloproteinase (MMP) family. Here we analyze the role of MMPs in the initiation of the mesenchymal cell phenotype in the developing heart, and find that they are necessary for the invasion of mesenchymal cells into the extracellular matrix of the endocardial cushion tissues. An important requirement in the formation of this mesenchyme is the turnover of type IV collagen along the basal surface of endocardial cells. In vitro experiments suggest that type IV collagen does not provide a suitable migratory substrate for endocardial cushion cells unless MMP-2 and MT-MMP are active. Relevant MMPs were found to be upregulated by factors known to be involved in the induction of the EMT such as TGFbeta3. These results provide evidence of an important role for MMPs during a specific stage of the epithelial mesenchymal transformation in the embryonic heart, and suggest that specific cell-matrix interactions which facilitate cell migration only occur when the composition of the surrounding extracellular matrix is proteolytically altered.     

Convective tissue movements play a major role in avian endocardial morphogenesis

Endocardial cells play a critical role in cardiac development and function, forming the innermost layer of the early (tubular) heart, separated from the myocardium by extracellular matrix (ECM). However, knowledge is limited regarding the interactions of cardiac progenitors and surrounding ECM during dramatic tissue rearrangements and concomitant cellular repositioning events that underlie endocardial morphogenesis. By analyzing the movements of immunolabeled ECM components (fibronectin, fibrillin-2) and TIE1 positive endocardial progenitors in time-lapse recordings of quail embryonic development, we demonstrate that the transformation of the primary heart field within the anterior lateral plate mesoderm (LPM) into a tubular heart involves the precise co-movement of primordial endocardial cells with the surrounding ECM. Thus, the ECM of the tubular heart contains filaments that were associated with the anterior LPM at earlier developmental stages. Moreover, endocardial cells exhibit surprisingly little directed active motility, that is, sustained directed movements relative to the surrounding ECM microenvironment. These findings point to the importance of large-scale tissue movements that convect cells to the appropriate positions during cardiac organogenesis.     

Endoglin is dispensable for angiogenesis, but required for endocardial cushion formation in the midgestation mouse embryo

Vascular patterning depends on precisely coordinated timing of endothelial cell differentiation and onset of cardiac function. Endoglin is a transmembrane receptor for members of the TGF-β superfamily that is expressed on endothelial cells from early embryonic gestation to adult life. Heterozygous loss of function mutations in human ENDOGLIN cause Hereditary Hemorrhagic Telangiectasia Type 1, a vascular disorder characterized by arteriovenous malformations that lead to hemorrhage and stroke. Endoglin null mice die in embryogenesis with numerous lesions in the cardiovascular tree including incomplete yolk sac vessel branching and remodeling, vessel dilation, hemorrhage and abnormal cardiac morphogenesis. Since defects in multiple cardiovascular tissues confound interpretations of these observations, we performed in vivo chimeric rescue analysis using Endoglin null embryonic stem cells. We demonstrate that Endoglin is required cell autonomously for endocardial to mesenchymal transition during formation of the endocardial cushions. Endoglin null cells contribute widely to endothelium in chimeric embryos rescued from cardiac development defects, indicating that Endoglin is dispensable for angiogenesis and vascular remodeling in the midgestation embryo, but is required for early patterning of the heart.     

Chick embryos exposed to trichloroethylene in an ex ovo culture model show selective defects in early endocardial cushion tissue formation

BACKGROUND: Formation of the primitive heart is a critical step for establishing a competent circulatory system necessary for continued morphogenesis, and as such has significant potential as a target for environmental insult. The goal of this study was to identify the initial cellular events that precede more superficially observable abnormalities resulting from exposing early chick embryos to trichloroethylene (TCE). METHODS: A whole embryo culture method was used to assess the susceptibility of endocardial epithelial-mesenchymal transformation in the early chick heart to TCE. This method has the benefits of maintaining the anatomical relationships of developing tissues and organs, instantaneously exposing precisely staged embryos to quantifiable levels of TCE in a protein-free medium, and the ability to directly monitor developmental morphology. RESULTS: A minority of embryos (Hamburger and Hamilton [HH] stage 13-14) exposed to TCE (10-80 ppm) were not viable after 24 hr in culture and exhibited a variety of gross malformations in a dose-dependent fashion. However, the majority of treated embryos remained viable and developed into HH stage 17 embryos that were superficially indistinguishable from vehicle-treated controls. Further analysis of the hearts of these superficially normal embryos by whole-mount confocal microscopy revealed selective reduction in the number of atrioventricular canal mesenchymal cells. Additionally, those mesenchymal cells that did develop migrated abnormally as long thin cords of adherent cells. CONCLUSIONS: The regional selectivity of these effects in the chick heart suggests a critical window of susceptibility to TCE in the epithelial-mesenchymal transformation of atrioventricular canal endocardium.     

Slug is a direct Notch target required for initiation of cardiac cushion cellularization

Snail family proteins are key regulators of epithelial-mesenchymal transition, but their role in endothelial-to-mesenchymal transition (EMT) is less well studied. We show that Slug, a Snail family member, is expressed by a subset of endothelial cells as well as mesenchymal cells of the atrioventricular canal and outflow tract during cardiac cushion morphogenesis. Slug deficiency results in impaired cellularization of the cardiac cushion at embryonic day (E)-9.5 but is compensated by increased Snail expression at E10.5, which restores cardiac cushion EMT. We further demonstrate that Slug, but not Snail, is directly up-regulated by Notch in endothelial cells and that Slug expression is required for Notch-mediated repression of the vascular endothelial cadherin promoter and for promoting migration of transformed endothelial cells. In contrast, transforming growth factor beta (TGF-beta) induces Snail but not Slug. Interestingly, activation of Notch in the context of TGF-beta stimulation results in synergistic up-regulation of Snail in endothelial cells. Collectively, our data suggest that combined expression of Slug and Snail is required for EMT in cardiac cushion morphogenesis.     

Genetic and cellular analyses of zebrafish atrioventricular cushion and valve development

Defects in cardiac valve morphogenesis and septation of the heart chambers constitute some of the most common human congenital abnormalities. Some of these defects originate from errors in atrioventricular (AV) endocardial cushion development. Although this process is being extensively studied in mouse and chick, the zebrafish system presents several advantages over these models, including the ability to carry out forward genetic screens and study vertebrate gene function at the single cell level. In this paper, we analyze the cellular and subcellular architecture of the zebrafish heart during stages of AV cushion and valve development and gain an unprecedented level of resolution into this process. We find that endocardial cells in the AV canal differentiate morphologically before the onset of epithelial to mesenchymal transformation, thereby defining a previously unappreciated step during AV valve formation. We use a combination of novel transgenic lines and fluorescent immunohistochemistry to analyze further the role of various genetic (Notch and Calcineurin signaling) and epigenetic (heart function) pathways in this process. In addition, from a large-scale forward genetic screen we identified 55 mutants, defining 48 different genes, that exhibit defects in discrete stages of AV cushion development. This collection of mutants provides a unique set of tools to further our understanding of the genetic basis of cell behavior and differentiation during AV valve development.     

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.