The bone morphogenetic protein antagonist noggin regulates mammalian cardiac morphogenesis

Bone morphogenetic proteins (BMPs) play many roles in mammalian cardiac development. Here we address the functions of Noggin, a dedicated BMP antagonist, in the developing mouse heart. In early cardiac tissues, the Noggin gene is mainly expressed in the myocardial cells of the outflow tract, atrioventricular canal, and future right ventricle. The major heart phenotypes of Noggin mutant embryos are thicker myocardium and larger endocardial cushions. Both defects result from increased cell number. Cell proliferation is increased and cell cycle exit is decreased in the myocardium. Although we find evidence of increased BMP signal transduction in the myocardium and endocardium, we show that the cardiac defects of Noggin mutants are rescued by halving the gene dosage of Bmp4. In culture, BMP increases the epithelial-to-mesenchymal transformation (EMT) of endocardial explant cells. Increased EMT likely accounts for the enlarged atrioventricular cushion. In the outflow tract cushion, we observed an increased contribution of cardiac neural crest cells to the mutant cushion mesenchyme, although many cells of the cushion were not derived from neural crest. Thus the enlarged outflow tract cushion of Noggin mutants likely arises by increased contributions both of endocardial cells that have undergone EMT as well as cells that have migrated from the neural crest. These data indicate that antagonism of BMP signaling by Noggin plays a critical role in ensuring proper levels of cell proliferation and EMT during cardiac morphogenesis in the mouse.     

Cell origins and tissue boundaries during outflow tract development

Morphogenesis of the cardiac outflow tract and aortic sac regions requires the progressive immigration and integrated differentiation of cells having very divergent embryonic histories. Mesodermal cells originating both within and beside the developing head contribute to endocardium and myocardium. These cells, together with later arriving neural crest cells, participate in the formation of the aorticopulmonary septum, truncal cushions, and semilunar valves, although there is uncertainty regarding the precise contributions of each. In addition, precursors of the enveloping epicardium and coronary arteries move into the outflow tract. Defining the spatial and temporal contributions of these disparate populations and the boundaries between them as the outflow tract shifts caudally is an essential prerequisite to understanding normal heart morphogenesis as well as the etiology of outflow tract dysmorphologies.     

Cardiac outflow tract malformations in chick embryos exposed to homocysteine

OBJECTIVE: Increased homocysteine concentrations have been associated with cardiac outflow tract defects. It has been hypothesized that cardiac neural crest cells were the target cells in these malformations. Cardiac neural crest cells migrate from the neural tube and contribute to the condensed mesenchyme of the aorticopulmonary septum and outflow tract cushions of the heart. The aim of this study is to investigate the effects of homocysteine on cardiac neural crest cells in relation to heart malformations. METHODS: Homocysteine was injected either into the neural tube lumen (30 micromol/l), or into the circulatory system (30 or 300 micromol/l) of chick embryos. LacZ-retroviral labeling was used to study cardiac neural crest cell migratory pathways after exposure to homocysteine. RESULTS: Cardiac neural crest cells contributed to the aorticopulmonary septum of both control and homocysteine-treated embryos. However, the outflow tract of homocysteine-neural tube injected embryos displayed 60% less apoptosis and 25% reduced myocardialization. A subarterial ventricular septal defect was observed in 83% of the embryos. None of these abnormalities were observed in homcysteine-circulatory system injected embryos. CONCLUSION: This study demonstrates that homocysteine disturbs apoptosis and myocardialization of the outflow tract, probably by affecting the cardiac neural crest cells.     

The homeobox gene Lbx1 specifies a subpopulation of cardiac neural crest necessary for normal heart development

Cardiac neural crest cells are known to play multiple roles during development of the inflow and outflow tract of the heart and the aortic arch. In addition, cardiac neural crest is required for normal heart tube looping and regulation of myocardial cell proliferation, as well as differentiation and function of the myocardium. We show that the homeobox gene Lbx1 is expressed in a subpopulation of the cardiac neural crest during tubular heart formation. Inactivation of the Lbx1 gene in mice resulted in defects in heart looping, changes in gene expression pattern, and increased cell proliferation ensuing in myocardial hyperplasia. We found that the activity of the Lbx1 promoter, as indicated by a LacZ reporter gene, is upregulated in the hearts of Lbx1(+/-):splotch(1H)/splotch(1H) and Lbx1(-/-) mice, indicating that Pax3 and Lbx1 participate in a negative regulatory feedback that might be necessary for normal differentiation and function of the myocardium during early heart development. Because migration of Lbx1-expressing neural crest cells was not altered in Lbx1(-/-) embryos, we postulate that Lbx1 gene function is critical for specification of a subpopulation of cardiac neural crest subsequent to migration.     

Pinch1 is required for normal development of cranial and cardiac neural crest-derived structures

Pinch1, an adaptor protein composed of 5 LIM domains, has been suggested to play an important role in multiple cellular processes. We found that Pinch1 is highly expressed in neural crest cells and their derivatives. To examine the requirement for Pinch1 in neural crest development, we generated neural crest conditional Pinch1 knockout mice using the Wnt1-Cre/loxP system. Neural crest conditional Pinch1 mutant embryos die perinatally from severe cardiovascular defects with an unusual aneurysmal common arterial trunk. Pinch1 mutants also exhibit multiple deficiencies in cranial neural crest-derived structures. Fate mapping demonstrated that initial migration of neural crest cells to the pharyngeal arch region occurs normally in the mutant embryos. However, in the cardiac outflow tract of mutants, neural crest cells exhibited hyperplasia and failed to differentiate into smooth muscle. Markedly increased apoptosis is observed in outflow tract cushions of mutants between embryonic days 11.5 and 13.5, likely contributing to the observed defects in cushion/valve remodeling and ventricular septation. Expression of transforming growth factor-beta(2), which plays a crucial role in outflow tract development, was decreased or absent in the outflow tract of the mutants. The decrease in transforming growth factor-beta(2) expression preceded neural crest cell death. Together, our results demonstrate that Pinch1 plays an essential role in neural crest development, perhaps in part through transforming growth factor-beta signaling.     

Genetic and developmental modulation of cardiac deficits in prenatal alcohol exposure

BACKGROUND: Increasing evidence demonstrates that genetic background is an important modulator of alcohol's effects on the developing fetus. Such effects are separable from maternal ethanol metabolism. Here, we study ethanol's effects on cardiogenesis in an avian model that shows strong cell death within neuronal and neural crest precursors following ethanol exposure. METHODS: The study design tested the hypothesis that ethanol-induced losses of cardiac neural crest populations would disrupt outflow tract development and thus contribute to the valvuloseptal deficits observed in prenatal alcohol exposure. Three chick strains were exposed to alcohol at gestational windows between gastrulation and early heart septation (day 3 incubation), and then hearts were examined at the completion of morphogenesis (day 10 incubation). RESULTS: Ethanol's impact on cardiac development was influenced by fetal genetics. The B300 x Hampshire Red cross exhibited pronounced cell death within cardiac neural crest populations but had normal development of the heart and aortic arches. Neural crest migration and differentiation into the distal outflow tract were also normal in these embryos, which suggested a capacity to repair earlier losses. The DeKalb White x Hampshire Red cross also did not exhibit cardiac defects. Hearts of the B300 strain had a unique phenotype with respect to ethanol exposure and exhibited a thin ventricular compact layer, dilatation, and reduced myosin/deoxyribonucleic acid and myosin/protein content, a phenotype that indicates disrupted myocardial maturation and inductive cues. The deficit was only observed when ethanol exposure occurred at stages 15 or 18 and apparently was independent of neural crest cell death. Such ventricular thinning might go undetected in the absence of extensive screening. CONCLUSIONS: Results add to the increasing evidence that genetic background strongly modulates the effects of prenatal alcohol exposure. The results also suggest that embryos have a varying capacity to repair and recover from earlier neural crest losses.     

Cellular components and signals required for the cardiac outflow tract assembly in Drosophila

Specification of cardiac primordia and formation of the Drosophila heart tube is highly reminiscent of the early steps of vertebrate heart development. We previously reported that the final morphogenesis of the Drosophila heart involves a group of nonmesodermal cells called heart-anchoring cells and a pair of derived from the pharyngeal mesoderm cardiac outflow muscles. Like the vertebrate cardiac neural crest cells, heart-anchoring cells migrate, interact with the tip of the heart, and participate in shaping the cardiac outflow tract. To better understand this process, we performed an in-depth analysis of how the Drosophila outflow tract is formed. We found that the most anterior cardioblasts that form a central outflow tract component, the funnel-shaped heart tip, do not originate from the cardiac primordium. They are initially associated with the pharyngeal cardiac outflow muscles and join the anterior aorta during outflow tract assembly. The particular morphology of the heart tip is disrupted in embryos in which heart-anchoring cells were ablated, revealing their critical role in outflow tract morphogenesis. We also demonstrate that Slit and Robo are required for directed movements of heart-anchoring cells toward the heart tip and that the cell-cell contact between the heart-anchoring cells and the ladybird-expressing cardioblasts is critically dependent on DE-cadherin Shotgun. Our observations suggest that the similarities between Drosophila and vertebrate cardiogenesis extend beyond the early developmental events.     

Notch signaling in cardiac development

The Notch signaling pathway has been demonstrated to play a critical role during mammalian cardiac development based on recent findings from gene-targeted mice. In addition, mutations in the Notch signaling pathway have been associated with human congenital heart defects such as Alagille syndrome, bicuspid aortic valve disease, calcification of the heart valves, and ventricular septal defects. Recently, it was demonstrated that Notch activation in the endocardium regulates ventricular myocardial development and that the Notch downstream target genes Hey1 and Hey2 are required for the establishment of the atrioventricular canal myocardial boundary. The Notch pathway has previously been implicated in regulating endothelial-to-mesenchymal transition during development of the heart valves, and recent reports further dissect the role of individual Notch downstream target genes during this process. In addition, a role for the Notch pathway during cardiac neural crest cell development has been identified, which provides a potential mechanism for the findings seen in Alagille syndrome. This review focuses on recently reported findings that elucidate mechanisms regulated by the Notch pathway during ventricular, atrioventricular canal, and outflow tract development.     

Cellular and molecular contributions of the cardiac neural crest to cardiovascular development

Normal development of the heart and great arteries requires participation of the cardiac neural crest. Ectomesenchymal cells from this area of the neural crest migrate to pharyngeal arches 3, 4, and 6, where they support development of the aortic arch arteries. Cells continue migration from the pharyngeal arches to specific sites in the outflow tract. Removal of the neural crest results in two types of malformations: outflow septation defects and alignment defects. The genesis of these two types of defects is by two different mechanisms. Outflow septation is disturbed when a critical number of cells does not reach the outflow tract. Alignment is altered by an as yet unknown secondary mechanism that is transmitted upstream to the heart from the pharyngeal arches. Aortic arch artery and ventricular development as well as hemodynamic parameters are abnormal from an early age. Some possible molecular mechanisms involved in specifying neural crest for participation in heart development are discussed.     

Hyperplastic conotruncal endocardial cushions and transposition of great arteries in perlecan-null mice

Perlecan is a heparan-sulfate proteoglycan abundantly expressed in pericellular matrices and basement membranes during development. Inactivation of the perlecan gene in mice is lethal at two developmental stages: around E10 and around birth. We report a high incidence of malformations of the cardiac outflow tract in perlecan-deficient embryos. Complete transposition of great arteries was diagnosed in 11 out of 15 late embryos studied (73%). Three of these 11 embryos also showed malformations of semilunar valves. Mesenchymal cells in the outflow tract were abnormally abundant in mutant embryos by E9.5, when the endocardial-mesenchymal transformation starts in wild-type embryos. At E10.5, mutant embryos lacked well-defined spiral endocardial ridges, and the excess of mesenchymal cells obstructed sometimes the outflow tract lumen. Most of this anomalous mesenchyme expressed the smooth muscle cell-specific alpha-actin isoform, a marker of the neural crest in the outflow tract of the mouse. In wild-type embryos, perlecan is present in the basal surface of myocardium and endocardium, as well as surrounding presumptive neural crest cells. We suggest that the excess of mesenchyme at the earlier stages of conotruncal development precludes the formation of the spiral ridges and the rotation of the septation complex in order to achieve a concordant ventriculoarterial connection. The observed mesenchymal overpopulation might be due to an uncontrolled migration of neural crest cells, which would arrive prematurely to the heart. Thus, perlecan is involved in the control of the outflow tract mesenchymal population size, underscoring the importance of the extracellular matrix in cardiac morphogenesis.     

Analysis of cranial neural crest distribution in the developing heart using quail-chick chimeras

Previous studies have shown that ablation of cranial neural crest results in heart malformations in chick embryos. Cranial neural crest cells populate all of the pharyngeal arches and provide the mesenchymal walls of the aortic arch arteries. Neural crest cells migrate from the pharyngeal apparatus into the outflow region of the heart. However, it is not known which of the pharyngeal arches contribute ectomesenchyme to the developing heart nor has a pattern of distribution in the outflow region been established. In the present study, premigratory presumptive arch neural crest from quail embryos was grafted homotopically onto early chick embryos. On Day 6 of incubation, the chimeric embryos were fixed and processed for histological evaluation. The neural crest providing mesenchyme to pharyngeal arches 1 and 2 was not associated with the developing heart. Neural crest presumptive for arches 3, 4, and 6 was found distributed to the outflow region of the heart. Neural crest from arch 4 contributed the largest number of cells to the developing aorticopulmonary and conotruncal septa. This information indicates that ablations of neural crest presumptive for arches 3, 4, and 6 influence heart development directly while lesions of other areas of cranial neural crest probably influence heart development only secondarily with the primary effects occurring in the pharyngeal arches.     

Mannheimer Lecture. The quintessence of the making of the heart

In my Mannheimer lecture, designed to meet the needs of a mainly clinical audience, I present aspects of cardiac development that link basic science to clinically relevant problems. During development of the cardiac tube, and its subsequent changes as a dextrally looped structure, which is still connected to the dorsal body wall by a venous and an arterial pole, there are basic requirements. These consist of the development of myocardium, endocardium and the interposed cardiac jelly from the cardiogenic plates. In this primitive heart tube, septation and valvar formation then take place to convert it into a four-chambered heart. I demonstrate that the refining of the above events cannot take place without the addition of extracardiac populations of cells. These are presented as the "quintessence of heart development", and consist of cells derived from the neural crest, along with epicardially derived cells. Without these contributions, the embryos uniformly die of cardiac insufficiency. Important contributions are made by the cells derived from the neural crest to septation and the formation of the arterial valves, and possibly in differentiation of the central conduction system. The epicardially derived cells are essential for formation of the interstitial fibroblasts and the myocardium, as well as the coronary vascular system. I conclude by discussing specific malformations of the heart that might be linked to these extracardiac contributions.     

Transforming growth factor-beta-induced differentiation of smooth muscle from a neural crest stem cell line

During vascular development, nascent endothelial networks are invested with a layer of supporting cells called pericytes in capillaries or smooth muscle in larger vessels. The cellular lineage of smooth muscle precursors and factors responsible for regulating their differentiation remain uncertain. In vivo, cells derived from the multipotent neural crest can give rise to vascular smooth muscle in parts of the head and also the cardiac outflow tract. Although transforming growth factor-beta (TGF-beta) has previously been shown to induce some smooth muscle markers from primary cultures of neural crest stem cells, the extent of the differentiation induced was not clear. In this study, we demonstrate that TGF-beta can induce many of the markers and characteristics of vascular smooth muscle from a neural crest stem cell line, Monc-1. Within 3 days of in vitro treatment, TGF-beta induces multiple smooth muscle-specific markers, while downregulating epithelial markers present on the parent cells. Treatment with TGF-beta also induces a contractile phenotype that responds to the muscarinic agonist carbachol and is not immediately reversed on TGF-beta withdrawal. Examination of the signaling pathways involved revealed that TGF-beta activation of Smad2 and Smad3 appear to be essential for the observed differentiation. Taken together, this system provides a novel model of smooth muscle differentiation that reliably recapitulates the process observed in vivo and allows for dissection of the pathways and processes involved in this process.     

Lineage and morphogenetic analysis of the cardiac valves

We used a genetic lineage-labeling system to establish the material contributions of the progeny of 3 specific cell types to the cardiac valves. Thus, we labeled irreversibly the myocardial (alphaMHC-Cre+), endocardial (Tie2-Cre+), and neural crest (Wnt1-Cre+) cells during development and assessed their eventual contribution to the definitive valvar complexes. The leaflets and tendinous cords of the mitral and tricuspid valves, the atrioventricular fibrous continuity, and the leaflets of the outflow tract valves were all found to be generated from mesenchyme derived from the endocardium, with no substantial contribution from cells of the myocardial and neural crest lineages. Analysis of chicken-quail chimeras revealed absence of any substantial contribution from proepicardially derived cells. Molecular and morphogenetic analysis revealed several new aspects of atrioventricular valvar formation. Marked similarities are seen during the formation of the mural leaflets of the mitral and tricuspid valves. These leaflets form by protrusion and growth of a sheet of atrioventricular myocardium into the ventricular lumen, with subsequent formation of valvar mesenchyme on its surface rather than by delamination of lateral cushions from the ventricular myocardial wall. The myocardial layer is subsequently removed by the process of apoptosis. In contrast, the aortic leaflet of the mitral valve, the septal leaflet of the tricuspid valve, and the atrioventricular fibrous continuity between these valves develop from the mesenchyme of the inferior and superior atrioventricular cushions. The tricuspid septal leaflet then delaminates from the muscular ventricular septum late in development.     

The effects of high phenylalanine concentration on chick embryonic development

Summary Cells from a particular portion of the cranial neural crest (cardiac neural crest) migrate from the neural fold into pharyngeal arches 3, 4 and 6, where they provide the support for the endothelium of the aortic arch arteries, and by migration into the outflow tract become involved in septation of the truncus arteriosus. Ablation of the premigratory cardiac neural crest results in persistent truncus arteriosus and other defects reminiscent of the DiGeorge syndrome in man. Removal of a small area of the cardiac neural crest causes a spectrum of heart defects classified together as dextraposed aorta including changes like that of Fallot's tetralogy in man. Some inflow tract anomalies have also been found. Pilot studies injecting phenylalanine into developing chick embryos at a very early stage had little effect on embryo viability or on the incidence of congenital heart defects. However, sham-treated animals produced predominantly small simple ventricular septal defects but phenylalanine-treated embryos had more serious and complex heart anomalies. It is not possible to say yet that congenital heart disease in the offspring of mothers with untreated phenylketonuria is due to phenylalanine-induced damage to the neural crest, but the pilot studies in chick suggest that this idea is worth pursuing.     

Functional insulin-like growth factor I receptors are expressed by neural-derived continuous cell lines

High affinity insulin-like growth factor I (IGF-I) receptors are expressed by two human neural derived cell lines, SK-N-SH and SK-N-MC. Specific [125I]IGF-I binding to crude membranes was 23.4% for SK-N-SH and 10.7% for SK-N-MC, with 50% inhibition of binding by unlabeled IGF-I between 0.6-0.7 nM. Scatchard analysis of crude membrane binding was linear, whereas Scatchard analysis after wheat germ agglutinin purification of the receptor became curvilinear. The IGF-I receptor alpha-subunits of SK-N-SH have an apparent Mr of 126K, whereas that for SK-N-MC is 132K. Despite these differences in alpha-subunit structure both cell lines demonstrate IGF-I-induced autophosphorylation of their own beta-subunits as well as specific IGF-I induced tyrosine kinase activity, suggesting normal coupling between the ligand-binding alpha-subunit and the tyrosine kinase-containing beta-subunit. Furthermore, IGF-I stimulated iododeoxyuridine uptake in both SK-N-SH and SK-N-MC in a dose-dependent manner, suggesting that these cells may be used to study the role of IGF-I action on neural tissues.     

Neuregulins with an Ig-like domain are essential for mouse myocardial and neuronal development.

Neuregulins are ligands for the erbB family of receptor tyrosine kinases and mediate growth and differentiation of neural crest, muscle, breast cancer, and Schwann cells. Neuregulins contain an epidermal growth factor-like domain located C-terminally to either an Ig-like domain or a cysteine-rich domain specific to the sensory and motor neuron-derived isoform. Here it is shown that elimination of the Ig-like domain-containing neuregulins by homologous recombination results in embryonic lethality associated with a deficiency of ventricular myocardial trabeculation and impairment of cranial ganglion development. The erbB receptors are expressed in myocardial cells and presumably mediate the neuregulin signal originating from endocardial cells. The trigeminal ganglion is reduced in size and lacks projections toward the brain stem and mandible. We conclude that IgL-domain-containing neuregulins play a major role in cardiac and neuronal development.