Harriett A. Stadt

HIRA, a DiGeorge Syndrome Candidate Gene, Is Required for Cardiac Outflow Tract Septation

DiGeorge syndrome (DGS) is a congenital disease characterized by defects in organs and tissues that depend on contributions by cell populations derived from neural crest for proper development. A number of candidate genes that lie within the q11 region of chromosome 22 commonly deleted in DGS patients have been identified. Orthologues of the DGS candidate gene HIRA are expressed in the neural crest and in neural crest-derived tissues in both chick and mouse embryos. By exposing a portion of the premigratory chick neural crest to phosphorothioate end-protected antisense oligonucleotides, ex ovo, followed by orthotopic backtransplantation to the untreated embryos, we have shown that the functional attenuation of cHIRA in the chick cardiac neural crest results in a significantly increased incidence of persistent truncus arteriosus, a phenotypic change characteristic of DGS, but does not affect the repatterning aortic arch arteries, the ventricular function, or the alignment of the outflow tract.

Hensen’s node gives rise to the ventral midline of the foregut: implications for organizing head and heart development

Patterning of the ventral head has been attributed to various cell populations, including endoderm, mesoderm, and neural crest. Here, we provide evidence that head and heart development may be influenced by a ventral midline endodermal cell population. We show that the ventral midline endoderm of the foregut is generated directly from the extreme rostral portion of Hensens node, the avian equivalent of the Spemann organizer. The endodermal cells extend caudally in the ventral midline from the prechordal plate during development of the foregut pocket. Thus, the prechordal plate appears as a mesendodermal pivot between the notochord and the ventral foregut midline. The elongating ventral midline endoderm delimits the right and left sides of the ventral foregut endoderm. Cells derived from the midline endoderm are incorporated into the endocardium and myocardium during closure of the foregut pocket and fusion of the bilateral heart primordia. Bilateral ablation of the endoderm flanking the midline at the level of the anterior intestinal portal leads to randomization of heart looping, suggesting that this endoderm is partitioned into right and left domains by the midline endoderm, thus performing a function similar to that of the notochord in maintaining left-right asymmetry. Because of its derivation from the dorsal organizer, its extent from the forebrain through the midline of the developing face and pharynx, and its participation in formation of a single midline heart tube, we propose that the ventral midline endoderm is ideally situated to function as a ventral organizer of the head and heart.

FGF8 signaling is chemotactic for cardiac neural crest cells

Cardiac neural crest cells migrate into the pharyngeal arches where they support development of the pharyngeal arch arteries. The pharyngeal endoderm and ectoderm both express high levels of FGF8. We hypothesized that FGF8 is chemotactic for cardiac crest cells. To begin testing this hypothesis, cardiac crest was explanted for migration assays under various conditions. Cardiac neural crest cells migrated more in response to FGF8. Single cell tracing indicated that this was not due to proliferation and subsequent transwell assays showed that the cells migrate toward an FGF8 source. The migratory response was mediated by FGF receptors (FGFR) 1 and 3 and MAPK/ERK intracellular signaling. To test whether FGF8 is chemokinetic and/or chemotactic in vivo, dominant negative FGFR1 was electroporated into the premigratory cardiac neural crest. Cells expressing the dominant negative receptor migrated slower than normal cardiac neural crest cells and were prone to remain in the vicinity of the neural tube and die. Treating with the FGFR1 inhibitor, SU5402 or an FGFR3 function-blocking antibody also slowed neural crest migration. FGF8 over-signaling enhanced neural crest migration. Neural crest cells migrated to an FGF8-soaked bead placed dorsal to the pharynx. Finally, an FGF8 producing plasmid was electroporated into an ectopic site in the ventral pharyngeal endoderm. The FGF8 producing cells attracted a thick layer of mesenchymal cells. DiI labeling of the neural crest as well as quail-to-chick neural crest chimeras showed that neural crest cells migrated to and around the ectopic site of FGF8 expression. These results showing that FGF8 is chemotactic and chemokinetic for cardiac neural crest adds another dimension to understanding the relationship of FGF8 and cardiac neural crest in cardiovascular defects.

Calcium influx through L-type CaV1.2 Ca2+ channels regulates mandibular development.

The identification of a gain-of-function mutation in CACNA1C as the cause of Timothy Syndrome (TS), a rare disorder characterized by cardiac arrhythmias and syndactyly, highlighted unexpected roles for the L-type voltage-gated Ca2+ channel CaV1.2 in nonexcitable cells. How abnormal Ca2+ influx through CaV1.2 underlies phenotypes such as the accompanying syndactyly or craniofacial abnormalities in the majority of affected individuals is not readily explained by established CaV1.2 roles. Here, we show that CaV1.2 is expressed in the first and second pharyngeal arches within the subset of cells that give rise to jaw primordia. Gain-of-function and loss-of-function studies in mouse, in concert with knockdown/rescue and pharmacological approaches in zebrafish, demonstrated that Ca2+ influx through CaV1.2 regulates jaw development. Cranial neural crest migration was unaffected by CaV1.2 knockdown, suggesting a role for CaV1.2 later in development. Focusing on the mandible, we observed that cellular hypertrophy and hyperplasia depended upon Ca2+ signals through CaV1.2, including those that activated the calcineurin signaling pathway. Together, these results provide new insights into the role of voltage-gated Ca2+ channels in nonexcitable cells during development.

Approaching Cardiac Development in Three Dimensions by Magnetic Resonance Microscopy

Cardiac neural crest (CNC) ablation in embryonic chicks leads to conotruncal anomalies of the heart as a result of altered cardiac looping. Altered looping results from failure of the myocardium from the secondary heart field to be added to the outflow tract. Various imaging techniques have been applied to visualize embryonic heart development. However, morphological abnormalities frequently cannot clearly be identified or appreciated in 2 dimensions, particularly those involving misorientation of cardiovascular structures and changes of myocardial volume. We present here 3-dimensional (3D) reconstructions of the embryonic chick heart at looping stages in sham-operated and CNC-ablated embryos acquired by magnetic resonance microscopy (MRM) using a new dual-contrast method for specimen preparation that combines perfusion fixation and immersion in fixative with a macro-molecular gadolinium-based contrast agent. In contrast to previous techniques, this method provides imaging not only of the cardiac chambers and vessel lumens but also of internal and external cardiac structures, such as the ventricular wall, myocardial trabeculations, cardiac jelly, and endocardial cushions. Furthermore, it allows morphovolumetric analysis of hearts at different stages. There is an excellent correlation between images obtained from MRM and those obtained by routine …

Compensatory responses and development of the nodose ganglion following ablation of placodal precursors in the embryonic chick (Gallus domesticus).

The nodose ganglion is the distal cranial ganglion of the vagus nerve which provides sensory innervation to the heart and other viscera. In this study, removal of the neuronal precursors which normally populate the right nodose ganglion was accomplished by ablating the right nodese placode in stage 9 chick embryos. Subsequent histological evaluation showed that in 54% of lesioned embryos surviving to day 6, the right ganglion was absent. Most embryos surviving to day 12, however, had identifiable right ganglia. In day 12 embryos, the right ganglion which developed was abnormal, with ganglion volume and ganglion cell diameter reduced by 50% and 20%, respectively, compared to control ganglia. To investigate the source of the neuron population in the regenerated ganglion, we combined nodose placode ablation with bilateral replacement of chick with quail “cardiac” neural crest (from mid-otic placode to somite 3). These cells normally provide only non-neuronal cells to the nodose ganglion, but produce neurons in other regions. At day 9, quail-derived neurons were identified in the right nodose ganglia of these chimeras, indicating that cardiac neural crest cells can generate neurons in the ganglion when placode-derived neurons are absent or reduced in number. On the other hand, we found that “sympathetic” neural crest (from somites 10 to 20) does not support ganglion development, suggesting that only neural crest cells normally present in the ganglion participate in reconstituting its neuronal population. Our previous work has shown that right nodose placode ablation produces abnormal cardiac function, which mimics a life-threatening human heart condition known as long QT syndrome. The present results suggest that the presence of neural crest-derived neurons in the developing right nodose ganglion may contribute to the functional abnormality in long QT syndrome.

Developmental Characteristics of the Chick Nodose Ganglion

Morphometric studies were carried out on the chick nodose ganglion between day 5 of incubation and 2 weeks after hatching. Previous findings showed that ablation of the nodose placode, the locus of precursor cells of nodose ganglion sensory neurons, results in abnormal cardiac function, and that these precursors can be induced to migrate abnormally to the heart and express abnormal phenotypes there, following cardiac neural crest ablation. These results prompted us to investigate further the normal development of nodose ganglion neurons. We find that the major period of neuron generation from placodal precursor cells in the ganglion occurs prior to day 5 of incubation. The loss of more than half of these neurons takes place between embryonic days 5 and 20, while neuron and ganglion sizes increase dramatically. Myelination is not seen at day 12 of incubation, but is present on day 15. Neurons continue to develop after hatching (day 21), reaching their adult size by 2 weeks after hatching. Unexpectedly, we found that the number of neurons increases after hatching, reaching the adult level of 62% more than embryonic day-20 numbers by 2 weeks after hatching. The mechanisms underlying the increase in neuron number after hatching are unexplained and require further investigation.

Membrane Ion Channels in Cardiac Malformation and Disease

Though nearly 1% of all live births are complicated by cardiovascular malformations,’ nothing is known concerning the electrophysiology of cardiac myocytes in response to congenital heart defects. This is due largely to the lack of a reliable experimental model from which electrophysiological measurements can be made at the cellular level. Based on present knowledge of electrical activity in myocytes from hearts in various adult experimental models of heart disease, some differences as a result of congenital malformation would be expected. Cardiac malformation would be expected to increase the hernodynamic burden of the heart muscle. According to Braunwald’ the mature heart basically depends on three mechanisms in order to compensate for an excessive hemodynamic burden and maintain cardiac output. These are (1) the Frank-Starling mechanism, (2) increased release of catecholamines by adrenergic nerves and the adrenal medulla, and ( 3) myocardial hypertrophy. Through the Frank-Starling mechanism the heart is intrinsically capable of varying its force of contraction on a beat-to-beat basis based on initial muscle (sarcomere) length. Catecholamines serve to increase muscle contractility and increase both Ca2+ and K’ conductances in the myocyte membrane, indirectly, through activation of betaadrenergic receptors. If the excessive hemodynamic burden is prolonged the heart will respond with hypertrophy of the myocardium. Hypertrophy represents an intrinsic change in the cardiac myocyte characterized by accelerated protein synthesis’ and, a t least initially, increased contractility.’ If the heart is not relieved of the excessive hemodynamic burden it will inevitably fail.2 Several changes in the electrical activity of myocytes have been associated with ventricular hypertrophy in a number of experimental models.6 The most consistent observation is a prolongation of the action potential that may be accompanied by a more depolarized diastolic potential and less membrane depolarization during the plateau p h a ~ e . ~ ” There are several underlying voltage and time-dependent Cazt and K + currents that may account for the prolonged action potential.“ Unfortunately, there have been just two voltage clamp studies on myocytes from hearts with experimentally induced hypertrophy. Using the sucrose gap technique in cat papillary muscle, Ten Eik and colleagues’ found both a reduction of the slow C a Z t current and a reduction of time-dependent outward K ’ in hearts with right ventricular