Atrioventricular cushion transformation is mediated by ALK2 in the developing mouse heart

Developmental abnormalities in endocardial cushions frequently contribute to congenital heart malformations including septal and valvular defects. While compelling evidence has been presented to demonstrate that members of the TGF-beta superfamily are capable of inducing endothelial-to-mesenchymal transdifferentiation in the atrioventricular canal, and thus play a key role in formation of endocardial cushions, the detailed signaling mechanisms of this important developmental process, especially in vivo, are still poorly known. Several type I receptors (ALKs) for members of the TGF-beta superfamily are expressed in the myocardium and endocardium of the developing heart, including the atrioventricular canal. However, analysis of their functional role during mammalian development has been significantly complicated by the fact that deletion of the type I receptors in mouse embryos often leads to early embryonal lethality. Here, we used the Cre/loxP system for endothelial-specific deletion of the type I receptor Alk2 in mouse embryos. The endothelial-specific Alk2 mutant mice display defects in atrioventricular septa and valves, which result from a failure of endocardial cells to appropriately transdifferentiate into the mesenchyme in the AV canal. Endocardial cells deficient in Alk2 demonstrate decreased expression of Msx1 and Snail, and reduced phosphorylation of BMP and TGF-beta Smads. Moreover, we show that endocardial cells lacking Alk2 fail to delaminate from AV canal explants. Collectively, these results indicate that the BMP type I receptor ALK2 in endothelial cells plays a critical non-redundant role in early phases of endocardial cushion formation during cardiac morphogenesis.     

Exposure of cardiac fibroblasts to the herbicide nitrofen causes altered interactions with the extracellular matrix

A large proportion of congenital heart defects result from dysmorphogenesis of valvuloseptal precursors, the endocardial cushions. Intrinsic to formation and maturation of these tissues are developmental changes in cell-cell and cell-extracellular matrix interactions. Interactions between cells and the extracellular matrix play critical roles in modulating cellular processes including proliferation, migration, differentiation and even survival. While significant progress is being made in the elucidation of the cellular events involved in valvuloseptal development, little is known regarding how environmental factors may affect this process. Embryonic exposure to the herbicide nitrofen has been shown to result in congenital heart defects associated with altered endocardial cushion formation or maturation. The present studies were performed to begin to address the cellular mechanisms of these nitrofen-induced effects. Heart fibroblasts were isolated and treated with varying doses of nitrofen in vitro. Experiments were performed to determine the effects of this herbicide on important cellular processes including migration, proliferation and apoptosis. These studies illustrated a dose-dependent decrease in collagen gel contraction and proliferation in response to nitrofen. Assays were also performed to determine the effects of nitrofen on fibroblast gene expression. Increased expression of collagen type I and specific integrins were seen following nitrofen exposure. These studies illustrate that nitrofen has direct effects on cardiac fibroblast proliferation and extracellular matrix remodeling, cellular events important in valvuloseptal development.     

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.     

Effects of vitamin A on endocardial cushion development in the mouse heart

Retinoic acid (RA) (78mg/kg) administered to ICR mice on days 9.0,9.5 and 10.0 of pregnancy (plug day = day 1), resulted in cardiac malformations in 37.6% of the surviving fetuses, including transposition of the great arteries, ventricular septal defects, and double outlet right ventricle. Histological examination of the hearts of embryos observed 24 hours after in vivo or in vitro exposure to RA on day 9 revealed abnormalities in endocardial cushion tissue. The volume of the atrioventricular endocardial was reduced in treated embryos as was the ratio of the size of the cushions to the size of the heart. The endothelial layer of the atrioventricular endocardial cushions appeared to be unaffected by the retinoic acid, however, the mesenchymal cushion cells were significantly reduced in number when compared with controls. Labeling with [3H]-thymidine indicated that the mitotic activity of the mesenchymal cell population was significantly decreased while that of the endothelial cells was comparable to control levels. The extracellular matrix or cardiac jelly of the endocardial cushions also appeared to be affected by RA exposure, as shown by studies utilizing colloidal iron to stain GAGs, which revealed a decrease in the amount of stainable material in treated cushions. Two possible cause for the reduced thymidine index of the cushion mesenchyme are discussed, namely, a direct effect of RA on the mesenchymal cells or an indirect effect via the altered extracellular matrix of the cushion tissue.     

Apoptosis as an instrument in cardiovascular development

Cell death as a phenomenon in embryonic development was first described over 100 years ago. Approximately 30 years ago the process was named apoptosis, and its involvement is now recognized in many life processes, in virtually every animal species, and from fertilization to the death of an organism. In cardiovascular development, it coincides with major developmental processes in specific time windows. Both intrinsic (controlled by mitochondrial activity) and extrinsic (starting with death receptors) apoptotic pathways co-regulate developmental mechanisms. During cardiac development, many cell populations are recruited to the heart, where they differentiate into cardiomyocytes, fibroblasts, smooth muscle cells, endocardial and endothelial cells lining the inner surfaces, and epicardial cells lining the outer contours. In particular, neural crest-derived cell populations, which migrate to specific locations in the heart, are prone to apoptosis. During the complex geometric changes that occur in the primary heart tube and connected vessel segments, proper interaction of the respective cell populations guarantees the ensuing steps of differentiation. Growth factors, including endothelin, VEGF, and TGF-beta, as well as other factors, such as FasL, play dominant roles in these phases. Transgenic and knockout studies have provided strong evidence for aberrant patterns of apoptosis resulting in congenital malformations and syndromic malformations, including septation anomalies, interrupted aortic arch segments, coronary anomalies, and DiGeorge syndrome. Embryonic remodeling of the arterial system, including the coronary arteries, is accompanied by apoptosis patterns, the disruption of which results in severe malformations. It is interesting to note that hemodynamic factors, such as flow-driven shear stress, regulate the expression of genes that are important for signaling molecules such as endothelin and NO-synthase. In general, high shear stress protects against apoptosis, thus preventing the onset of disease processes in the fully-grown vasculature, and regulating the remodeling of the vascular system in the embryo.     

A novel role for VEGF in endocardial cushion formation and its potential contribution to congenital heart defects

Normal cardiovascular development is exquisitely dependent on the correct dosage of the angiogenic growth factor and vascular morphogen vascular endothelial growth factor (VEGF). However, cardiac expression of VEGF is also robustly augmented during hypoxic insults, potentially mediating the well-established teratogenic effects of hypoxia on heart development. We report that during normal heart morphogenesis VEGF is specifically upregulated in the atrioventricular (AV) field of the heart tube soon after the onset of endocardial cushion formation (i.e. the endocardium-derived structures that build the heart septa and valves). To model hypoxia-dependent induction of VEGF in vivo, we conditionally induced VEGF expression in the myocardium using a tetracycline-regulated transgenic system. Premature induction of myocardial VEGF in E9.5 embryos to levels comparable with those induced by hypoxia prevented formation of endocardial cushions. When added to explanted embryonic AV tissue, VEGF fully inhibited endocardial-to-mesenchymal transformation. Transformation was also abrogated in AV explants subjected to experimental hypoxia but fully restored in the presence of an inhibitory soluble VEGF receptor 1 chimeric protein. Together, these results suggest a novel developmental role for VEGF as a negative regulator of endocardial-to-mesenchymal transformation that underlies the formation of endocardial cushions. Moreover, ischemia-induced VEGF may be the molecular link between hypoxia and congenital defects in heart septation.     

Timeline and distribution of melanocyte precursors in the mouse heart

Apart from the well-studied melanocytes of the skin, eye and inner ear, another population has recently been described in the heart. In this study, we tracked cardiac melanoblasts using in situ hybridization with a dopachrome tautomerase (Dct) probe and Dct-LacZ transgenic mice. Large numbers of melanoblasts were found in the atrioventricular (AV) endocardial cushions at embryonic day (E) 14.5 and persisted in the AV valves into adulthood. The earliest time Dct-LacZ-positive cells were observed in the AV endocardial cushions was E12.5. Prior to that, between E10.5 and E11.5, small numbers of melanoblasts traveled between the post-otic area and third somite along the anterior and common cardinal veins and branchial arch arteries with other neural crest cells expressing CRABPI. Cardiac melanocytes were not found in the spotting mutants Ednrb s-l/s-l and Kit w-v/w-v, while large numbers were observed in transgenic mice that overexpress endothelin 3. These results indicate that cardiac melanocytes depend on the same signaling molecules known to be required for proper skin melanocyte development and may originate from the same precursor population. Cardiac melanocytes were not found in zebrafish or frog but were present in quail suggesting an association between cardiac melanocytes and four-chambered hearts.     

Generation of Smad7‐Cre recombinase mice: A useful tool for the study of epithelial‐mesenchymal transformation within the embryonic heart

Smad7 can be induced by various transforming growth factor‐β superfamily ligands and negatively modulates their signaling, thus acting in a negative, autocrine feedback manner. Previous analyses have demonstrated that although Smad7 is widely expressed, it is predominantly found in the vascular endothelium. Because of the restricted spatiotemporal reporter expression driven via a novel 4.3 kb Smad7 promoter in endocardial cells overlying the hearts atrioventricular (AV) cushions; we hypothesized that a transgenic Cre line would prove useful for the analysis of endocardial cushion and valve formation. Here we describe a mouse line, Smad7^Cre, where Cre is robustly expressed within both cardiac outflow and AV endocardial cushions. Additionally, as endocardial cells are thought to contribute at least in part to the formation of the endocardial cushion mesenchyme, we crossed the Smad7^Cre mice to the ROSA26^eGFP‐DTA diphtheria toxin A‐expressing mice in order to genetically ablate Smad7^Cre expressing cells. Ablation of Smad7^Cre cells resulted in embryonic lethality by E11.5 and largely acellular endocardial cushions. genesis 47:469–475, 2009. © 2009 Wiley‐Liss, Inc.     

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.     

Apoptotic force: active mechanical function of cell death during morphogenesis

Apoptosis, or programmed cell death, is an essential process for the elimination of unnecessary cells during embryonic development, tissue homeostasis, and certain pathological conditions. Recently, an active mechanical function of apoptosis called apoptotic force has been demonstrated during a tissue fusion process of Drosophila embryogenesis. The mechanical force produced during apoptosis is used not only to force dying cells out from tissues in order to keep tissue integrity, but also to change the morphology of neighboring cells to fill the space originally occupied by the dying cell. Furthermore, the occurrence of apoptosis correlates with tissue movement and tension of the tissue. This finding suggests that apoptotic forces might be harnessed throughout cell death-related morphogenesis; however, this concept remains to be fully investigated. While the investigation of this active mechanical function of apoptosis has just begun, here we summarize the current understandings of this novel function of apoptosis, and discuss some possible developmental processes in which apoptosis may play a mechanical role. The concept of apoptotic force prompts a necessity to rethink the role of programmed cell death during morphogenesis.     

Tbx20 regulation of endocardial cushion cell proliferation and extracellular matrix gene expression

While recent work has implicated Tbx20 in myocardial maturation and proliferation, the role of Tbx20 in heart valve development remains relatively unknown. Tbx20 expression was manipulated in primary avian endocardial cells in order to elucidate its function in developing endocardial cushions. Tbx20 gain of function was achieved with a Tbx20-adenovirus, and endogenous Tbx20 expression was inhibited with Tbx20-specific siRNA in cultured endocardial cushion cells. With Tbx20 gain of function, the expression of chondroitin sulfate proteoglycans (CSPG), including aggrecan and versican, was decreased, while the expression of the matrix metalloproteinases (MMP) mmp9 and mmp13 was increased. Consistent results were observed with Tbx20 loss of function, where the expression of CSPG genes increased and MMP genes decreased. In addition, cushion mesenchyme proliferation increased with infection of a Tbx20-adenovirus and decreased with transfection of Tbx20-specfic siRNA. Furthermore, BMP2 treatment resulted in increased Tbx20 expression in endocardial cushion cells, and loss of Tbx20 led to increased Tbx2 and decreased N-myc gene expression. Taken together, these data support a role for Tbx20 in repressing extracellular matrix remodeling and promoting cell proliferation in mesenchymal valve precursor populations in endocardial cushions during embryonic development.     

Notch1b and neuregulin are required for specification of central cardiac conduction tissue

Normal heart function is critically dependent on the timing and coordination provided by a complex network of specialized cells: the cardiac conduction system. We have employed functional assays in zebrafish to explore early steps in the patterning of the conduction system that previously have been inaccessible. We demonstrate that a ring of atrioventricular conduction tissue develops at 40 hours post-fertilization in the zebrafish heart. Analysis of the mutant cloche reveals a requirement for endocardial signals in the formation of this tissue. The differentiation of these specialized cells, unlike that of adjacent endocardial cushions and valves, is not dependent on blood flow or cardiac contraction. Finally, both neuregulin and notch1b are necessary for the development of atrioventricular conduction tissue. These results are the first demonstration of the endocardial signals required for patterning central ;slow' conduction tissue, and they reveal the operation of distinct local endocardial-myocardial interactions within the developing heart tube.     

Fibroblast growth factor (FGF)-4 can induce proliferation of cardiac cushion mesenchymal cells during early valve leaflet formation

While much has been learned about how endothelial cells transform to mesenchyme during cardiac cushion formation, there remain fundamental questions about the developmental fate of cushions. In the present work, we focus on the growth and development of cushion mesenchyme. We hypothesize that proliferative expansion and distal elongation of cushion mesenchyme mediated by growth factors are the basis of early valve leaflet formation. As a first step to test this hypothesis, we have localized fibroblast growth factor (FGF)-4 protein in cushion mesenchymal cells at the onset of prevalve leaflet formation in chick embryos (Hamburger and Hamilton stage 20-25). Ligand distribution was correlated with FGF receptor (FGFR) expression. In situ hybridization data indicated that FGFR3 mRNA was confined to the endocardial rim of the atrioventricular (AV) cushion pads, whereas FGFR2 was expressed exclusively in cushion mesenchymal cells. FGFR1 expression was detected in both endocardium and cushion mesenchyme as well as in myocardium. To determine whether the FGF pathways play regulatory roles in cushion mesenchymal cell proliferation and elongation into prevalvular structure, FGF-4 protein was added to the cushion mesenchymal cells explanted from stage 24-25 chick embryos. A significant increase in proliferative ability was strongly suggested in FGF-4-treated mesenchymal cells as judged by the incorporation of 5'-bromodeoxyuridine (BrdU). To determine whether cushion cells responded similarly in vivo, a replication-defective retrovirus encoding FGF-4 with the reporter, bacterial beta-galactosidase was microinjected into stage 18 chick cardiac cushion mesenchyme along the inner curvature where AV and outflow cushions converge. As compared with vector controls, overexpression of FGF-4 clearly induced expansion of cushion mesenchyme toward the lumen. To further test the proliferative effect of FGF-4 in cardiac cushion expansion in vivo (ovo), FGF-4 protein was microinjected into stage 18 chick inner curvature. An assay for BrdU incorporation indicated a significant increase in proliferative ability in FGF-4 microinjected cardiac cushion mesenchyme as compared with BSA-microinjected controls. Together, these results suggest a role of FGF-4 for cardiac valve leaflet formation through proliferative expansion of cushion mesenchyme.