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 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.     

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.     

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.     

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.     

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.     

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.     

Serotonin regulates mouse cranial neural crest migration.

Serotonergic agents (uptake inhibitors, receptor ligands) cause significant craniofacial malformations in cultured mouse embryos suggesting that 5-hydroxytryptamine (serotonin) (5-HT) may be an important regulator of craniofacial development. To determine whether serotonergic regulation of cell migration might underly some of these effects, cranial neural crest (NC) explants from embryonic day 9 (E9) (plug day = E1) mouse embryos or dissociated mandibular mesenchyme cells (derived from NC) from E12 embryos were placed in a modified Boyden chamber to measure effects of serotonergic agents on cell migration. A dose-dependent effect of 5-HT on the migration of highly motile cranial NC cells was demonstrated, such that low concentrations of 5-HT stimulated migration, whereas this effect was progressively lost as the dose of 5-HT was increased. In contrast, most concentrations of 5-HT inhibited migration of less motile, mandibular mesenchyme cells. To investigate the possible involvement of specific 5-HT receptors in the stimulation of NC migration, several 5-HT subtype-selective antagonists were used to block the effects of the most stimulatory dose of 5-HT (0.01 microM). Only NAN-190 (a 5-HT1A antagonist) inhibited the effect of 5-HT, suggesting involvement of this receptor. Further evidence was obtained by using immunohistochemistry with 5-HT receptor antibodies, which revealed expression of the 5-HT1A receptor but not other subtypes by migrating NC cells in both embryos and cranial NC explants. These results suggest that by activating appropriate receptors 5-HT may regulate migration of cranial NC cells and their mesenchymal derivatives in the mouse embryo.     

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.     

Development of teeth in chick embryos after mouse neural crest transplantations

Teeth were lost in birds 70-80 million years ago. Current thinking holds that it is the avian cranial neural crest-derived mesenchyme that has lost odontogenic capacity, whereas the oral epithelium retains the signaling properties required to induce odontogenesis. To investigate the odontogenic capacity of ectomesenchyme, we have used neural tube transplantations from mice to chick embryos to replace the chick neural crest cell populations with mouse neural crest cells. The mouse/chick chimeras obtained show evidence of tooth formation showing that avian oral epithelium is able to induce a nonavian developmental program in mouse neural crest-derived mesenchymal cells.     

Relation of early hemodynamic changes to final cardiac phenotype and survival after neural crest ablation in chick embryos

BACKGROUND. Microcinephotography was used to study a model of persistent truncus arteriosus created in chick embryos by ablation of premigratory neural crest destined for the third and fourth aortic arch arteries as well as the septum of the cardiac outflow tract. METHODS AND RESULTS. Twenty-five control embryos and 105 of 202 experimental embryos were filmed on day 3 of incubation and then reincubated. The remaining 97 experimental embryos were not filmed because of twisting of the embryos, but they were reincubated. There was no difference in either the survival rate (p greater than 0.23) from day 3 to day 11 of incubation or the incidence of persistent truncus arteriosus (p greater than 0.08) between the filmed and the nonfilmed embryos. Incomplete looping of the cardiac tube observed in experimental embryos during early cardiogenesis correlated with a right ventricular origin of the outflow vessels in the definitive heart. Hemodynamic measurements indicated that there was no difference in heart rate, ejection fraction, systolic and diastolic areas, stroke volume, and cardiac output between controls and the experimental group as a whole. However, embryos that did not survive to day 11 had decreased stroke volume (p less than 0.001) and cardiac output (p less than 0.001), whereas embryos that survived to day 11 with cardiac malformations had increased stroke volume and cardiac output in early embryogenesis. CONCLUSIONS. Increased stroke volume and cardiac output may be necessary factors for survival in embryos with cardiac dysmorphogenesis and probably are associated with dilation of the ventricular portion of the cardiac tube, which leads to malalignment of the outflow vessel or vessels.     

Sox9 is required for determination of the chondrogenic cell lineage in the cranial neural crest

Sox9 has essential roles in endochondral bone formation during axial and appendicular skeletogenesis. Sox9 is also expressed in neural crest cells, but its function in neural crest remains largely unknown. Because many craniofacial skeletal elements are derived from cranial neural crest (CNC) cells, we asked whether deletion of Sox9 in CNC cells by using the Cre recombinase/loxP recombination system would affect craniofacial development. Inactivation of Sox9 in neural crest resulted in a complete absence of cartilages and endochondral bones derived from the CNC. In contrast, all of the mesodermal skeletal elements and intramembranous bones were essentially conserved. The migration and the localization of Sox9-null mutant CNC cells were normal. Indeed, the size of branchial arches and the frontonasal mass of mutant embryos was comparable to that of WT embryos, and the pattern of expression of Ap2, a marker of migrating CNC cells, was normal. Moreover, in mouse embryo chimeras Sox9-null mutant cells migrated to their correct location in endochondral skeletal elements; however, Sox9-null CNC cells were unable to contribute chondrogenic mesenchymal condensations. In mutant embryos, ectopic expression of osteoblast marker genes, such as Runx2, Osterix, and Col1a1, was found in the locations where the nasal cartilages exist in WT embryos. These results indicate that inactivation of Sox9 causes CNC cells to lose their chondrogenic potential. We hypothesize that these cells change their cell fate and acquire the ability to differentiate into osteoblasts. We conclude that Sox9 is required for the determination of the chondrogenic lineage in CNC cells.     

Apoptosis is not required for mammalian neural tube closure

Apoptotic cell death occurs in many tissues during embryonic development and appears to be essential for processes including digit formation and cardiac outflow tract remodeling. Studies in the chick suggest a requirement for apoptosis during neurulation, because inhibition of caspase activity was found to prevent neural tube closure. In mice, excessive apoptosis occurs in association with failure of neural tube closure in several genetic mutants, but whether regulated apoptosis is also necessary for neural tube closure in mammals is unknown. Here we investigate the possible role of apoptotic cell death during mouse neural tube closure. We confirm the presence of apoptosis in the neural tube before and during closure, and identify a correlation with 3 main events: bending and fusion of the neural folds, postfusion remodeling of the dorsal neural tube and surface ectoderm, and emigration of neural crest cells. Both Casp3 and Apaf1 null embryos exhibit severely reduced apoptosis, yet neurulation proceeds normally in the forebrain and spine. In contrast, the mutant embryos fail to complete neural tube closure in the midbrain and hindbrain. Application of the apoptosis inhibitors z-Vad-fmk and pifithrin-α to neurulation-stage embryos in culture suppresses apoptosis but does not prevent initiation or progression of neural tube closure along the entire neuraxis, including the midbrain and hindbrain. Remodeling of the surface ectoderm to cover the closed tube, as well as delamination and migration of neural crest cells, also appear to be normal in the apoptosis-suppressed embryos. We conclude that apoptosis is not required for neural tube closure in the mouse embryo.     

Decreased neural crest stem cell expansion is responsible for the conotruncal heart defects within the splotch (Sp(2H))/Pax3 mouse mutant

OBJECTIVE: Several mouse models of cardiac neural crest cell (NCC)-associated conotruncal heart defects exist, but the specific cellular and molecular defects within cardiac NCC morphogenesis remain largely unknown. Our objective was to investigate the underlying mechanisms resulting in outflow tract defects and why insufficient cardiac NCC reach the heart of the Splotch (Sp(2H)) mouse mutant embryo. METHODS: For this study we used in vitro cell culture techniques, in vivo mouse-chick chimeras, BrdU cell proliferation labeling, TUNEL labeling to visualize apoptosis and the molecular markers AP-2, Wnt-1 and Wnt-3a to characterize NCC morphogenesis in vivo. RESULTS: Expression of the NCC marker AP-2 revealed an extensive reduction in migratory NCC, however the rates of cell proliferation and apoptosis were unaffected, and do not account for the Sp(2H) NCC-associated heart defects. Further expression analysis revealed that Wnt-1, but not Wnt-3a, is expressed at decreased levels within Sp(2H) and that the cardiac NCC fail to undergo normal NC stem cell proliferative expansion prior to migration while still in the neural folds. However, when placed into a wild-type matrix or a tissue culture environment, the Sp(2H) cardiac NCC could migrate normally. Additionally, this reduced population of Sp(2H) NC stem cells do migrate properly within the Sp(2H) environment, as observed by neurofilament expression and cardiac innervation. CONCLUSIONS: Taken together, all these data indicate that the Sp(2H) defect is intrinsic to the NC stem cells themselves and that there is a decrease in the number of pre-migratory cardiac NCC that form. It appears that this decrease in NCC number is the primary defect that ultimately leads to a lack of a cardiac NCC-derived Sp(2H) outflow tract septum.     

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.     

Pathogenesis of various forms of double outlet right ventricle in mouse fetal trisomy 13.

The pathogenesis of double outlet right ventricle with or without pulmonary infundibular atresia in mouse fetal trisomy 13 was studied at the organ level using microdissection and scanning electron microscopy. Altogether, 394 karyotyped trisomic embryos were collected between 11 days and 16 hours of gestation (presence of a vaginal plug = day 1) and 15 days of gestation at intervals of 8 hours, and at 16 days of gestation. The hearts were perfusion-fixed, microdissected, and prepared to be observed in scanning electron microscope in the following standardized orientations: frontal, right or left profile, septal and parietal halves of the right ventricle and outflow tract (conotruncus). Comparison of 276 trisomic hearts with their normal counterparts described previously has shown that: the first pathognomonic feature is the abnormal anterior position of the proximal part of the parietal outflow tract ridge or of both ridges (at 12 days and 16 hours of gestation); the abnormal anterior fusion of these ridges ("coalescence") results in a mesenchymal mass behind which is deviated the pulmonary part of the outflow tract lumen; from 14 days and 16 hours of gestation on, this lumen is either obstructed, resulting in a supravalvar stenosis of the pulmonary trunk and subsequently evolving into double outlet right ventricle with pulmonary infundibular atresia; or, in a minority of cases, this lumen is not obstructed and the heart develops into double outlet right ventricle without pulmonary infundibular atresia. The pathogenesis of these malformations differs from most of the known hypotheses based on deductions from human malformed hearts, as well as from observations of the pathogenesis of similar outflow tract malformations, such as those found in the Keeshond dog or rats treated with trimethadione.     

Increased Cell Death and Reduced Neural Crest Cell Numbers in Ethanol-Exposed Embryos: Partial Basis for the Fetal Alcohol Syndrome Phenotype

Fetal alcohol syndrome (FAS) is characterized by growth retardation, craniofacial malformations, and heart and neural defects; the cellular and molecular mechanism(s) responsible for ethanol's teratogenicity remains unknown. Although the phenotype suggests that prenatal ethanol exposure perturbs neural crest cell development, direct proof that these cells are an in utero target is still lacking. Previous research suggested that cranial neural crest cells are eliminated by ethanol-induced apoptosis. We tested this hypothesis using a chick embryo model of FAS. A single dose of ethanol, chosen to achieve a concentration of 35–42 mg/dl, was injected in ovo at gastrulation and resulted in growth retardation, craniofacial foreshortening, and disrupted hindbrain segmentation. Ethanol exposure enhanced cell death within areas populated by cranial neural crest cells, particularly in the hindbrain and craniofacial mesenchyme. In contrast, control embryos had limited cell death within these regions. Subsequent immunolabeling with neural crest cell-specific antibody revealed that ethanol treatment resulted in fewer neural crest cell numbers, whereas neural crest migration patterns were unaffected by ethanol. These results suggest that prenatal ethanol exposure leads to loss of cranial neural crest cells. Such a loss could result, in part, in the phenotype characteristic of FAS.