Sox9 expression and ultrastructure of the endocardial cushions during the heart development in Xenopus laevís

The etiology of ethanol-related congenital heart defects has been the focus of much study, but most research has concentrated on cellular and molecular mechanisms. We have shown with optical coherence tomography (OCT) that ethanol exposure led to increased retrograde flow and smaller atrioventricular (AV) cushions compared to controls. Since AV cushions play a role in patterning the conduction delay at the atrioventricular junction (AVJ), this study aims to investigate whether ethanol exposure alters the AVJ conduction in early looping hearts and whether this alteration is related to the decreased cushion size. Quail embryos were exposed to a single dose of ethanol at gastrulation, and Hamburger-Hamilton stage 19 - 20 hearts were dissected for imaging. Cardiac conduction was measured using an optical mapping microscope and we imaged the endocardial cushions using OCT. Our results showed that, compared with controls, ethanol-exposed embryos exhibited abnormally fast AVJ conduction and reduced cushion size. However, this increased conduction velocity (CV) did not strictly correlate with decreased cushion volume and thickness. By matching the CV map to the cushion size map, we found that the slowest conduction location was consistently at the atrial side of the AVJ, which had the thinner cushions, not at the thickest cushion location at the ventricular side as expected. Our findings reveal regional differences in the AVJ myocardium even at this early stage in heart development. These findings reveal the early steps leading to the heterogeneity and complexity of conduction at the mature AVJ, a site where arrhythmias can be initiated.

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From cushions to leaflets: morphogenesis of cardiac atrioventricular valves

10.1093/med/9780198757269.003.0017 ◽

2018 ◽

Author(s):

Donal MacGrogan ◽

José Maria Pérez-Pomares ◽

Bill Chaudhry ◽

José Luis de la Pompa ◽

Deborah J. Henderson

Keyword(s):

Heart Development ◽

Cellular Proliferation ◽

Valve Disease ◽

Signalling Pathways ◽

Valve Leaflet ◽

Endocardial Cushions ◽

Developmental Defects ◽

Mesenchymal Transition ◽

Valve Development ◽

Developmental Signalling

At the looping stage of heart development, tissue patterning of myocardium and endocardium at the atrioventricular (AV) junction defines a morphogenic field competent to form valves that initially appear as protrusions of proteoglycan-rich extracellular matrix (ECM) called endocardial cushions (ECs) which are cellularized by an endocardial-mesenchymal transition (EMT). Cellular proliferation results in fusion of the major AV mesenchymal cushions and AV septation, whereas smaller cushions receive a supply from epicardially derived cells. These various sources of mesenchyme precursors give rise to most of the valve structures, leaflets, annuli, and supporting tension apparatus. During valve leaflet maturation, the ECM matrix accumulates collagen and elastin and assembles into a thin flexible fibrous structure, which is remarkably tough. Valve development is regulated by the cross-talk between developmental signalling pathways. Pathogenic mutations in a subset of developmentally important genes have been linked to valve disease, suggesting that developmental defects may underlie valve disease in adulthood.

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Regulation of apoptosis in the endocardial cushions of the developing chick heart

AJP Cell Physiology ◽

10.1152/ajpcell.00509.2001 ◽

2002 ◽

Vol 282 (6) ◽

pp. C1348-C1360 ◽

Cited By ~ 13

Author(s):

William M. Keyes ◽

Esmond J. Sanders

Keyword(s):

Heart Development ◽

Time Course ◽

Nuclear Staining ◽

Caspase 9 ◽

Endocardial Cushions ◽

Similar Time ◽

Cleavage Fragment ◽

Protein Levels ◽

Developing Heart

During the early stages of heart development, there are two main foci of cell death: outflow tract (OT) and atrioventricular (AV) endocardial cushions. These tissues contribute to the septa and valves of the mature heart and receive cell populations from neural crest (NC) cell migration and epicardial cell invasion. We examined embryonic chick hearts for expression, in the cushions, of bcl-2 family members, caspase-9, and the caspase substrate poly(ADP-ribose) polymerase. Antiapoptotic bcl-2 is expressed heavily in the OT and AV regions throughout embryonic days (ED) 4–7, with a decrease in levels at ED 4 and 5 in OT and AV cushions, respectively. Proapoptotic bax predominantly associated with the prongs of the NC-derived aorticopulmonary (AP) septum but was expressed throughout the AV cushions. Proapoptotic bak also associated with the prongs of the AP septum in the OT, while protein levels were upregulated at ED 4–5 and 4–6 in OT and AV cushions, respectively. Bid expression showed a similar time course. We found the 10-kDa cleavage fragment of active caspase-9 at ED 4–8 and 5–8 in OT and AV cushions, respectively, and the 24-kDa cleavage fragment of poly(ADP-ribose) polymerase throughout ED 3–8 and 7–8 in OT and AV cushions, respectively. Caspase-3 cleavage occurred throughout the time period examined. Using cushion cell cultures, we found that inhibitors of caspases-3 and -9 and a universal caspase inhibitor significantly reduced apoptosis, as did retroviral overexpression of bcl-2 using an RCAS expression vector. Premigratory NC cells were fluorescently labeled in vivo with 1,1-didodecyl-3,3,3′,3′-tetramethylindocarbocyanine. Subsequent nuclear staining of cushion cells with 4,6-diamidino-2-phenylindole revealed the presence of apoptotic nuclei in the NC cells in the OT cushions and in the prongs of the AP septum. These results demonstrate a developmentally regulated role for the bcl-2 and the caspase families of molecules in the endocardial cushions of the developing heart and lend support to the possibility that some of the dying cells in the cushions are derived from the NC.

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β-Catenin is required for endothelial-mesenchymal transformation during heart cushion development in the mouse

The Journal of Cell Biology ◽

10.1083/jcb.200403050 ◽

2004 ◽

Vol 166 (3) ◽

pp. 359-367 ◽

Cited By ~ 269

Author(s):

Stefan Liebner ◽

Anna Cattelino ◽

Radiosa Gallini ◽

Noemi Rudini ◽

Monica Iurlaro ◽

...

Keyword(s):

Wnt Signaling ◽

Heart Development ◽

Transcriptional Activity ◽

Ex Vivo ◽

Smooth Muscle Actin ◽

Muscle Actin ◽

Endocardial Cushions ◽

Smad Phosphorylation ◽

Mesenchymal Transformation

During heart development endocardial cells within the atrio-ventricular (AV) region undergo TGFβ-dependent epithelial-mesenchymal transformation (EMT) and invade the underlying cardiac jelly. This process gives rise to the endocardial cushions from which AV valves and part of the septum originate. In this paper we show that in mouse embryos and in AV explants TGFβ induction of endocardial EMT is strongly inhibited in mice deficient for endothelial β-catenin, leading to a lack of heart cushion formation. Using a Wnt-signaling reporter mouse strain, we demonstrated in vivo and ex vivo that EMT in heart cushion is accompanied by activation of β-catenin/TCF/Lef transcriptional activity. In cultured endothelial cells, TGFβ2 induces α-smooth muscle actin (αSMA) expression. This process was strongly reduced in β-catenin null cells, although TGFβ2 induced smad phosphorylation was unchanged. These data demonstrate an involvement of β-catenin/TCF/Lef transcriptional activity in heart cushion formation, and suggest an interaction between TGFβ and Wnt-signaling pathways in the induction of endothelial-mesenchymal transformation.

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Mutation in the Trapα/Ssr1 Gene, Encoding Translocon-Associated Protein α, Results in Outflow Tract Morphogenetic Defects

Molecular and Cellular Biology ◽

10.1128/mcb.00913-06 ◽

2006 ◽

Vol 26 (20) ◽

pp. 7760-7771 ◽

Cited By ~ 15

Author(s):

K. Mesbah ◽

A. Camus ◽

C. Babinet ◽

J. Barra

Keyword(s):

Heart Development ◽

Proximal Part ◽

Double Outlet Right Ventricle ◽

Outflow Tract ◽

Endoplasmic Reticulum Membrane ◽

Messenger Rnas ◽

Endocardial Cushions ◽

Gene Encoding ◽

Mutant Cells

ABSTRACT Translocon-associated protein complex (TRAP) is thought to be required for efficient protein-specific translocation across the endoplasmic reticulum membrane. We created a mutation in the Trapα gene that leads to the synthesis of a truncated TRAPα protein fused to ShBle-β-galactosidase. Analysis of Trapα cDNAs reveals that among three different messenger RNAs expressed in the mouse, one of them encodes a slightly larger protein that differs in its C-terminal end. This mRNA, specific for skeletal muscle and heart, is only expressed after birth. Homozygous Trapα mutant pups die at birth, likely as a result of severe cardiac defects. Indeed, the septation of the proximal part of the outflow tract is absent, resulting in a double-outlet right ventricle. Studies of protein secretion in transfected embryonic fibroblasts reveal that the TRAP complex does not function properly in homozygous mutant cells and confirm, in vivo, the involvement of TRAP in substrate-specific translocation. Our results provide the first in vivo demonstration that a member of the TRAP complex plays a crucial role in mammalian heart development and suggest that TRAPα could be involved in translocation of factors necessary for maturation of endocardial cushions.

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Confocal Imaging of Early Heart Development in Xenopus laevis

Developmental Biology ◽

10.1006/dbio.1999.9558 ◽

2000 ◽

Vol 218 (1) ◽

pp. 64-73 ◽

Cited By ~ 65

Author(s):

Sandra J Kolker ◽

Urszula Tajchman ◽

Daniel L Weeks

Keyword(s):

Xenopus Laevis ◽

Heart Development ◽

Confocal Imaging

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Cardiac myosin heavy chain expression during heart development in Xenopus laevis

Differentiation ◽

10.1046/j.1432-0436.1995.5840269.x ◽

1995 ◽

Vol 58 (4) ◽

pp. 269-280 ◽

Cited By ~ 2

Author(s):

William G. Cox ◽

Anton W. Neff

Keyword(s):

Xenopus Laevis ◽

Myosin Heavy Chain ◽

Heavy Chain ◽

Heart Development ◽

Cardiac Myosin

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

Development ◽

10.1242/dev.128.9.1531 ◽

2001 ◽

Vol 128 (9) ◽

pp. 1531-1538 ◽

Cited By ~ 5

Author(s):

Y. Dor ◽

T.D. Camenisch ◽

A. Itin ◽

G.I. Fishman ◽

J.A. McDonald ◽

...

Keyword(s):

Growth Factor ◽

Heart Development ◽

Heart Defects ◽

Negative Regulator ◽

Congenital Defects ◽

Endocardial Cushion ◽

Potential Contribution ◽

Vegf Receptor ◽

Endocardial Cushions ◽

Mesenchymal Transformation

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.

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Lack of endothelial cell survivin causes embryonic defects in angiogenesis, cardiogenesis, and neural tube closure

Blood ◽

10.1182/blood-2006-06-028068 ◽

2007 ◽

Vol 109 (11) ◽

pp. 4742-4752 ◽

Cited By ~ 47

Author(s):

Femke Zwerts ◽

Florea Lupu ◽

Astrid De Vriese ◽

Saskia Pollefeyt ◽

Lieve Moons ◽

...

Keyword(s):

Endothelial Cell ◽

Neural Tube ◽

Heart Development ◽

Neural Tube Closure ◽

Endocardial Cushions ◽

Vascular Integrity ◽

Apoptosis Protein ◽

Mesenchymal Transformation ◽

Tube Closure

Abstract We explored the physiologic role of endothelial cell apoptosis during development by generating mouse embryos lacking the inhibitor of apoptosis protein (IAP) survivin in endothelium. This was accomplished by intercrossing survivinlox/lox mice with mice expressing cre recombinase under the control of the endothelial cell specific tie1 promoter (tie1-cre mice). Lack of endothelial cell survivin resulted in embryonic lethality. Mutant embryos had prominent and diffuse hemorrhages from embryonic day 9.5 (E9.5) and died before E13.5. Heart development was strikingly abnormal. Survivin-null endocardial lineage cells could not support normal epithelial-mesenchymal transformation (EMT), resulting in hypoplastic endocardial cushions and in utero heart failure. In addition, 30% of mutant embryos had neural tube closure defects (NTDs) that were not caused by bleeding or growth retardation, but were likely due to alterations in the release of soluble factors from endothelial cells that otherwise support neural stem cell proliferation and neurulation. Thus, regulation of endothelial cell survival, and maintenance of vascular integrity by survivin are crucial for normal embryonic angiogenesis, cardiogenesis, and neurogenesis.

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The Morphology of Heart Development in Xenopus laevis

Developmental Biology ◽

10.1006/dbio.1999.9559 ◽

2000 ◽

Vol 218 (1) ◽

pp. 74-88 ◽

Cited By ~ 81

Author(s):

Timothy J. Mohun ◽

Li Ming Leong ◽

Wolfgang J. Weninger ◽

Duncan B. Sparrow

Keyword(s):

Xenopus Laevis ◽

Heart Development

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