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Ryk is a member of the receptor tyrosine kinase (RTK) family of proteins that control and regulate cellular processes. It is distinguished by binding Wnt ligands and having no detectable intrinsic protein tyrosine kinase activity suggesting Ryk is a pseudokinase. Here, we show an essential role for Ryk in directing morphogenetic events required for normal cardiac development through the examination of Ryk-deficient mice. We employed vascular corrosion casting, vascular perfusion with contrast dye, and immunohistochemistry to characterize cardiovascular and pharyngeal defects in Ryk-/- embryos. Ryk-/- mice exhibit a variety of malformations of the heart and outflow tract that resemble human congenital heart defects. This included stenosis and interruption of the aortic arch, ventriculoarterial malalignment, ventricular septal defects and abnormal pharyngeal arch artery remodelling. This study therefore defines a key intersection between a subset of growth factor receptors involved in planar cell polarity signalling, the Wnt family and mammalian cardiovascular development.

The cardiac neural crest (CNC) plays pivotal roles in numerous steps of cardiac development. Every aspect of the CNC cell’s lifespan is highly orchestrated, from its induction in the dorsal neural tube to its migration to its differentiation at its final destination. During migration, CNC cells are affected by their environment and simultaneously modulate the extra-cellular milieu through which they migrate. In the pharyngeal arches, CNC cells repattern the originally symmetrical arch arteries, producing the great arteries. Because the cardiac neural crest is essential for many aspects of heart development, it is unsurprising that human CNC-related syndromes have severe phenotypes. This chapter describes how CNC cells are formed and contribute to their final destinations. Essential signalling pathways are presented in the context of CNC development, and CNC-related syndromes are included to highlight this population’s broad importance during 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.

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.

The myocardium of the heart is composed of multiple highly specialized myocardial lineages, including those of the ventricular and atrial myocardium, and the specialized conduction system. Specification and maturation of each of these lineages during heart development is a highly ordered, ongoing process involving multiple signaling pathways and their intersection with transcriptional regulatory networks. Here, we attempt to summarize and compare much of what we know about specification and maturation of myocardial lineages from studies in several different vertebrate model systems. To date, most research has focused on early specification, and although there is still more to learn about early specification, less is known about factors that promote subsequent maturation of myocardial lineages required to build the functioning adult heart.

Cardiac neural crest cells originate as part of the postotic caudal rhombencephalic neural crest stream. Ectomesenchymal cells in this stream migrate to the circumpharyngeal ridge and then into the caudal pharyngeal arches where they condense to form first a sheath and then the smooth muscle tunics of the persisting pharyngeal arch arteries. A subset of the cells continue migrating into the cardiac outflow tract where they will condense to form the aorticopulmonary septum. Cell signaling, extracellular matrix and cell-cell contacts are all critical for the initial migration, pauses, continued migration, and condensation of these cells. This review elucidates what is currently known about these factors.

During heart development, a subpopulation of cells in the heart field maintains cardiac potential over several days of development and forms the myocardium and smooth muscle of the arterial pole. Using clonal and explant culture experiments, we show that these cells are a stem cell population that can differentiate into myocardium, smooth muscle and endothelial cells. The multipotent stem cells proliferate or differentiate into different cardiovascular cell fates through activation or inhibition of FGF and BMP signaling pathways. BMP promoted myocardial differentiation but not proliferation. FGF signaling promoted proliferation and induced smooth muscle differentiation, but inhibited myocardial differentiation. Blocking the Ras/Erk intracellular pathway promoted myocardial differentiation, while the PLCγ and PI3K pathways regulated proliferation. In vivo, inhibition of both pathways resulted in predictable arterial pole defects. These studies suggest that myocardial differentiation of arterial pole progenitors requires BMP signaling combined with downregulation of the FGF/Ras/Erk pathway. The FGF pathway maintains the pool of proliferating stem cells and later promotes smooth muscle differentiation.

Aortic valve atresia with interruption of the aortic arch is an extremely rare anomaly; only eleven cases of this anomaly have been reported to date. In the absence of additional sources of blood flow to the ascending aorta, aortic valve atresia with interruption of the aortic arch is fatal. We present, to the best of our knowledge, the first case of a live birth with aortic valve atresia and interrupted left aortic arch (type B) without evidence of an aorticopulmonary communication or ductal supply to the native ascending aorta. Instead, blood flow to the native aortic root was derived from a persistent right embryonic dorsal aorta.

Cardiac neural crest cells (CNCC) migrate into the caudal pharynx and arterial pole of the heart to form the outflow septum. Ablation of the CNCC results in arterial pole malalignment and failure of outflow septation, resulting in a common trunk overriding the right ventricle. Unlike preotic cranial crest, the postotic CNCC do not normally regenerate. We applied the hedgehog signaling inhibitor, cyclopamine (Cyc), to chick embryos after CNCC ablation and found normal heart development at day 9 suggesting that the CNCC population was reconstituted. We ablated the CNCC, and labeled the remaining neural tube with DiI/CSRE and applied cyclopamine. Cells migrated from the neural tube in the CNCC-ablated, cyclopamine-treated embryos but not in untreated CNCC-ablated embryos. The newly generated cells followed the CNCC migration pathways, expressed neural crest markers and supported normal heart development. Finally, we tested whether reducing hedgehog signaling caused redeployment of the dorsal–ventral axis of the injured neural tube, allowing generation of new neural crest-like cells. The dorsal neural tube marker, Pax7, was maintained 12 h after CNCC ablation with Cyc treatment but not in the CNCC-ablated alone. This disruption of dorsal–ventral neural patterning permits a new wave of migratory cardiac neural crest-like cells.

The Sonic hedgehog (Shh)-null mouse was initially described as a phenotypic mimic of Tetralogy of Fallot with pulmonary atresia (Washington Smoak, I., Byrd, N.A., Abu-Issa, R., Goddeeris, M.M., Anderson, R., Morris, J., Yamamura, K., Klingensmith, J., and Meyers, E.N. 2005. Sonic hedgehog is required for cardiac outflow tract and neural crest cell development. Dev. Biol. 283, 357–372.); however, subsequent reports describe only a single outflow tract, leaving the phenotype and its developmental mechanism unclear. We hypothesized that the phenotype that occurs in response to Shh knockdown is pulmonary atresia and is directly related to the abnormal development of the secondary heart field. We found that Shh was expressed by the pharyngeal endoderm adjacent to the secondary heart field and that its receptor Ptc2 was expressed in a gradient in the secondary heart field, with the most robust expression in the caudal secondary heart field, closest to the Shh expression. In vitro culture of secondary heart field with the hedgehog inhibitor cyclopamine significantly reduced proliferation. In ovo, cyclopamine treatment before the secondary heart field adds to the outflow tract reduced proliferation only in the caudal secondary heart field, which coincided with the region of high Ptc2 expression. After outflow tract septation should occur, embryos treated with cyclopamine exhibited pulmonary atresia, pulmonary stenosis, and persistent truncus arteriosus. In hearts with pulmonary atresia, cardiac neural crest-derived cells, which form the outflow tract septum, migrated into the outflow tract and formed a septum. However, this septum divided the outflow tract into two unequal sized vessels and effectively closed off the pulmonary outlet. These experiments show that Shh is necessary for secondary heart field proliferation, which is required for normal pulmonary trunk formation, and that embryos with pulmonary atresia have an outflow tract septum.

Retroviral and transgenic lineage-tracing studies have shown that neural crest cells associate with the developing bundles of the ventricular conduction system. Whereas this migration of cells does not provide progenitors for the myocardial cells of the conduction system, the question of whether neural crest affects the differentiation and/or function of cardiac specialized tissues continues to be of interest. Using optical mapping of voltage-sensitive dye, we determined that ventricles from chick embryos in which the cardiac neural crest had been laser ablated did not progress to apex-to-base activation by the expected stage [i.e., Hamburger and Hamilton (HH) 35] but instead maintained basal breakthroughs of epicardial activation consistent with immature function of the conduction system. In direct studies of activation, waves of depolarization originating from the His bundle were found to be uncommon in control hearts from HH34 and HH35 embryos. However, activations propagating from septal base, at or near the His bundle, occurred frequently in hearts from HH34 and HH35 neural crest-ablated embryos. Consistent with His bundle cells maintaining electrical connections with adjacent working myocytes, histological analyses of hearts from neural crest-ablated embryos revealed His bundles that had not differentiated a lamellar organization or undergone a process of compaction and separation from surrounding myocardium observed in controls. Furthermore, measurements on histological sections from optically mapped hearts indicated that, whereas His bundle diameter in control embryos thinned by almost one-half between HH30 and HH34, the His bundle in ablated embryos underwent no such compaction in diameter, maintaining a thickness at HH30, HH32, and HH34 similar to that observed in HH30 controls. We conclude that the cardiac neural crest is required in a novel function involving lamellar compaction and electrical isolation of the basally located His bundle from surrounding myocardium.

Neural crest cells are multipotential cells that delaminate from the dorsal neural tube and migrate widely throughout the body. A subregion of the cranial neural crest originating between the otocyst and somite 3 has been called "cardiac neural crest" because of the importance of these cells in heart development. Much of what we know about the contribution and function of the cardiac neural crest in cardiovascular development has been learned in the chick embryo using quail-chick chimeras to study neural crest migration and derivatives as well as using ablation of premigratory neural crest cells to study their function. These studies show that cardiac neural crest cells are absolutely required to form the aorticopulmonary septum dividing the cardiac arterial pole into systemic and pulmonary circulations. They support the normal development and patterning of derivatives of the caudal pharyngeal arches and pouches, including the great arteries and the thymus, thyroid and parathyroids. Recently, cardiac neural crest cells have been shown to modulate signaling in the pharynx during the lengthening of the outflow tract by the secondary heart field. Most of the genes associated with cardiac neural crest function have been identified using mouse models. These studies show that the neural crest cells may not be the direct cause of abnormal cardiovascular development but they are a major component in the complex tissue interactions in the caudal pharynx and outflow tract. Since, cardiac neural crest cells span from the caudal pharynx into the outflow tract, they are especially susceptible to any perturbation in or by other cells in these regions. Thus, understanding congenital cardiac outflow malformations in human sequences of malformations as represented by the DiGeorge syndrome will necessarily require understanding development of the cardiac neural crest.

Abnormal patterning of coronary arteries (CAs) is a clinically significant problem, and as yet, few animal models have been systematically investigated for coronary patterning defects. Here we characterized coronary artery (CA) insertion and branching patterns of the proximal coronary stems in the hearts of wildtype and heterozygous connexin43 knockout (Cx431 KO) mice. This study entailed the use of high-resolution micro CT imaging for three-dimensional coronary reconstructions. MicroCT of 17 wildtype mice showed a remarkably consistent pattern of CA deployment in the normal mouse heart. Two main CA stems are inserted from the left and right into the aorta. The right coronary artery then branches immediately into the right main and the septal-conal branch, while the left coronary artery branches further distally into the circumflex and the anterior descending CA. This patterning of CA anatomy was confirmed by histology, and by using a vascular smooth muscle or endothelial cell specific lacZ reporter gene to delineate the CAs. A parallel analysis of 25 heterozygous Cx431 KO mouse hearts showed 22 had defects in patterning of the CAs. They exhibited a wide variation in CA anatomy, including abnormal origin and course of the main CA stems, multiple accessories, and dual septal-conal branches. Overall, these studies show loss of one Cx431 allele (haploinsufficiency) leads to a high incidence of coronary patterning defects. These findings suggest CA patterning is sensitive to Cx431 gene dosage. Developmental Dynamics 235:2786 –2794, 2006. †Published 2006 Wiley-Liss, Inc.

The arterial pole of the heart is the region where the ventricular myocardium continues as the vascular smooth muscle tunics of the aorta and pulmonary trunk. It has been shown that the arterial pole myocardium derives from the secondary heart field and the smooth muscle tunic of the aorta and pulmonary trunk derives from neural crest. However, this neural crest-derived smooth muscle does not extend to the arterial pole myocardium leaving a region at the base of the aorta and pulmonary trunk that is invested by vascular smooth muscle of unknown origin. Using tissue marking and vascular smooth muscle markers, we show that the secondary heart field, in addition to providing myocardium to the cardiac outflow tract, also generates prospective smooth muscle that forms the proximal walls of the aorta and pulmonary trunk. As a result, there are two seams in the arterial pole: first, the myocardial junction with secondary heart field-derived smooth muscle; second, the secondary heart field-derived smooth muscle with the neural crest-derived smooth muscle. Both of these seams are points where aortic dissection frequently occurs in Marfan's and other syndromes.

Die meisten kongenitalen Anomalien des Herzens haben ihren Ursprung in und wahrend der fruhen Morphogenese, und selbst einige Herzerkrankungen, die erst im Erwachsenenalter auftreten, entstehen schon wahrend der embryologischen Entwicklung des Herzens. Konotrunkale Herzfehler umfassen eine grose Kategorie von angeborenen Defekten und kommen bei Kindern in relativ hoher Haufigkeit vor. Diese Herzfehler, die sowohl in utero als auch postnatal nach herzchirurgischen Eingriffen mit einem hohen Letalitatsrisiko einhergehen, sind entwicklungsgeschichtlich haufig mit Dextroposition der Aorta verbunden, welche sich in Anomalien des ventrikularen Ausflusstraktes in Form von Fehlausrichtung (Malalignment) der grosen Gefase wiederfindet. Die Atiologie und Pathogenese der Mechanismen, die zu einer dextroponierten Aorta fuhren, sind nicht bekannt. Es wird aber angenommen, dass diese Fehlstellung auf eine gestorte Herzschleifenbildung und/oder fehlerhaftes „Wedging“ des Ausflusstraktes (d. h. Verkeilung der Aorta zwischen den Atrioventrikularklappen) wahrend der fruhen Herzentwicklung zuruckzufuhren ist. Wir haben in einer Serie von Studien die Morphologie und visuelle Entwicklung des embryonalen Herzens in einem experimentellen Model von dextroponierter Aorta untersucht, um mogliche Mechanismen der Dextropostion der Aorta zu eruieren. Hierbei wurden neben zahlreichen etablierten Methoden zur Analyse der Anatomie und Pathologie (Morphologische Analysen, Kardiale Morphometrie, Histologie, Elektronenmikroskopie und Immunhistochemie) auch neue bildgebende Verfahren (Videocinephotographie und Zeitraffer–Studien mittels einer digitalen Hochgeschwindigkeitskamera, Konfokale und Raster–Elektronenmikroskopie, Optische Koharenztomographie (OCT) und Magnetresonanzmikroskopie (MRM) zur 3–D–Rekonstruktion des Herzens) angewandt, um die normalen und pathologischen Veranderungen wahrend der Herzentwicklung besser zu visualisieren. Die vorliegende Schrift fasst die Ergebnisse dieser Studien zusammen.

Tbx2 belongs to a family of developmental transcription regulatory factors. We evaluated whether the gap junction protein Connexin43 (Cx43), an important regulator of osteoblast function and bone development, may be a downstream target gene regulated by Tbx2. The Cx43 promoter contains direct repeats of the consensus T-box binding motif, TCACAC, and moreover, Tbx2 and Cx43 show overlapping expression domains in precursors to bone and in osteoblasts. In vitro analysis showed that the Cx43 promoter contains two Tbx2 binding sites, and this binding was dependent on the TCACAC consensus sequence. Transient transfection analysis with a Cx43 promoter-driven lacZ reporter construct revealed negative regulation mediated by these two Tbx2 binding sites in osteoblast-like cells. Thus, downregulation of Tbx2 led to de-repression of wild-type Cx43 promoter activity, whereas a promoter construct with mutated binding sites showed no de-repression. In stably transfected osteosarcoma cells in which expression of the endogenous Tbx2 gene was downregulated with a Tbx2 antisense construct, a marked de-repression of the endogenous Cx43 gene was observed. This was accompanied by a marked increase in the abundance of Cx43 gap junctions and increased functional gap junction-mediated cell-cell communication. Analysis of lacZ expression in transgenic mice containing the mutated Cx43 promoter-driven lacZ construct further suggested de-repression of the Cx43 promoter in limb buds, a region destined to give rise to long bones of the limbs. Taken together, these findings indicate that the promoter of Cx43 is repressible by Tbx2, both in cultured osteoblast-like cells in vitro and likely in the developing embryo.

Myocardial dysfunction is evident within hours after ablation of the cardiac neural crest in chick embryos, suggesting a role for neural crest in myocardial maturation that is separate from its role in outflow septation. This role could be conserved in an animal that does not have a divided systemic and pulmonary circulation, such as zebrafish. To test this hypothesis, we used cell marking to identify the axial level of neural crest that migrates to the heart in zebrafish embryos. Unlike the chick and mouse, the zebrafish cardiac neural crest does not originate from the axial level of the somites. The region of neural crest cranial to somite 1 was found to contribute cells to the heart. Cells from the cardiac neural crest migrated to the myocardial wall of the heart tube, where some of them expressed a myocardial phenotype. Laser ablation of the cardiac premigratory neural crest at the three- to four-somite stage resulted in loss of the neural crest cells migrating to the heart as shown by the absence of AP2- and HNK1-expressing cells and failure of the heart tube to undergo looping. Myocardial function was assessed 24 hr after the cardiac neural crest ablation in a subpopulation of embryos with normal heart rate. Decreased stroke volume, ejection fraction, and cardiac output were observed, indicating a more severe functional deficit in cardiac neural crest-ablated zebrafish embryos compared with neural crest-ablated chick embryos. These results suggest a new role for cardiac neural crest cells in vertebrate cardiac development and are the first report of a myocardial cell lineage for neural crest derivatives.

Background Twenty years ago this year was the first publication describing a region of neural crest cells necessary for normal cardiovascular development. Ablation of this region in chick resulted in persistent truncus arteriosus, mispatterning of the great vessels, outflow malalignments, and hypoplasia or aplasia of the pharyngeal glands. Methods We begin with a historical perspective and then review the progress that has been made in the ensuing 20 years in determining the direct and indirect contributions of the neural crest cells, now termed cardiac neural crest cells, in cardiovascular and pharyngeal arch development. Many of the molecular pathways that are now known to influence the specification, migration, patterning and final targeting of the cardiac neural crest cells are also reviewed. Results Although much knowledge has been gained by using many genetic manipulations to understand the cardiac neural crest cells' role in cardiovascular development, most models fail to explain the phenotypes seen in syndromic and non-syndromic human congenital heart defects, such as the DiGeorge syndrome. Conclusions We propose that the cardiac neural crest exists as part of a larger cardiocraniofacial morphogenetic field and describe several human syndromes that result from abnormal development of this field. Birth Defects Research (Part C) 69:2–13, 2003. © 2003 Wiley-Liss, Inc.

Background— Optical coherence tomography (OCT) is a depth-resolved, noninvasive, non-destructive imaging modality, the use of which has yet to be fully realized in developmental biology. Methods and Results— We visualized embryonic chick hearts at looping stages using an OCT system with a 22 μm axial and 27 μm lateral resolution and an acquisition rate of 4000 A-scans per second. Normal chick embryos from stages 14 to 22 and sham-operated and cardiac neural crest-ablated embryos from stages 15 and 18 were scanned by OCT. Three-dimensional data sets were acquired and processed to create volumetric reconstructions and short video clips. The OCT-scanned embryos (2 in each group) were photographed after histological sectioning in comparable planes to those visualized by OCT. The optical and histological results showing cardiovascular microstructures such as myocardium, the cardiac jelly, and endocardium are presented. Conclusions— OCT is a powerful imaging modality which can provide new insight in assessing and understanding normal and abnormal cardiac development in a variety of animal models.

Development of the heart is a complex process involving primary and secondary heart fields that are set aside to generate myocardial and endocardial cell lineages. The molecular inductions that occur in the primary heart field appear to be recapitulated in induction and myocardial differentiation of the secondary heart field, which adds the conotruncal segments to the primary heart tube. While much is now known about the initial steps and factors involved in induction of myocardial differentiation, little is known about induction of endocardial development. Many of the genes expressed by nascent myocardial cells, which then become committed to a specific heart segment, have been identified and studied. In addition to the heart fields, several other “extracardiac” cell populations contribute to the fully functional mature heart. Less is known about the genetic programs of extracardiac cells as they enter the heart and take part in cardiogenesis. The molecular/genetic basis of many congenital cardiac defects has been elucidated in recent years as a result of new insights into the molecular control of developmental events.

In utero interventions aimed at restoring left ventricular hemodynamic forces in fetuses with prenatally diagnosed hypoplastic left heart syndrome failed to stimulate ventricular myocardial growth during gestation, suggesting chamber growth during development may not rely upon fluid forces. We therefore hypothesized that ventricular hypertrophy during development may depend upon fundamental Ca2 +-dependent growth pathways that function independent of hemodynamic forces. To test this hypothesis, zebrafish embryos were treated with inhibitors or activators of Ca2 + signaling in the presence or absence of contraction during the period of chamber development. Abolishment of contractile function alone in the setting of preserved Ca2 + signaling did not impair ventricular hypertrophy. In contrast, inhibition of L-type voltage-gated Ca2 + influx abolished contraction and led to reduced ventricular hypertrophy, whereas increasing L-type voltage-gated Ca2 + influx led to enhanced ventricular hypertrophy in either the presence or absence of contraction. Similarly, inhibition of the downstream Ca2 +-sensitive phosphatase calcineurin, a known regulator of adult cardiac hypertrophy, led to reduced ventricular hypertrophy in the presence or absence of contraction, whereas hypertrophy was rescued in the absence of L-type voltage-gated Ca2 + influx and contraction by expression of a constitutively active calcineurin. These data suggest that ventricular cardiomyocyte hypertrophy during chamber formation is dependent upon Ca2 + signaling pathways that are unaffected by heart function or hemodynamic forces. Disruption of Ca2 +-dependent hypertrophy during heart development may therefore represent one mechanism for impaired chamber formation that is not related to impaired blood flow.

Point mutations in the calcium-permeable TRPV4 ion channel have been identified as the cause of autosomal-dominant human motor neuropathies, arthropathies, and skeletal malformations of varying severity. The objective of this study was to determine the mechanism by which TRPV4 channelopathy mutations cause skeletal dysplasia. The human TRPV4V620I channelopathy mutation was transfected into primary porcine chondrocytes and caused significant (2.6-fold) up-regulation of follistatin (FST) expression levels. Pore altering mutations that prevent calcium influx through the channel prevented significant FST up-regulation (1.1-fold). We generated a mouse model of theTRPV4V620I mutation, and found significant skeletal deformities (e.g., shortening of tibiae and digits, similar to the human disease brachyolmia) and increases in Fst/TRPV4 mRNA levels (2.8-fold). FST was significantly up-regulated in primary chondrocytes transfected with 3 different dysplasia-causing TRPV4 mutations (2- to 2.3-fold), but was not affected by an arthropathy mutation (1.1-fold). Furthermore, FST-loaded microbeads decreased bone ossification in developing chick femora (6%) and tibiae (11%). FST gene and protein levels were also increased 4-fold in human chondrocytes from an individual natively expressing the TRPV4T89I mutation. Taken together, these data strongly support that up-regulation of FST in chondrocytes by skeletal dysplasia-inducing TRPV4 mutations contributes to disease pathogenesis.—Leddy, H. A., McNulty, A. L., Lee, S. H., Rothfusz, N. E., Gloss, B., Kirby, M. L., Hutson, M. R., Cohn, D. H., Guilak, F., Liedtke, W. Follistatin in chondrocytes: the link between TRPV4 channelopathies and skeletal malformations.

Ablation of premigratory cardiac neural crest results in defective development of the cardiac outflow tract. The purpose of the present study was to correlate the earliest functional and morphological changes in heart development after cardiac neural crest ablation. Within 24 hours after neural crest ablation, the external morphology of the hearts showed straight outflow limbs, tighter heart loops, and variable dilations. Incorporation of bromodeoxyuridine in myocytes, an indication of proliferation, was doubled after cardiac neural crest ablation. The myocardial calcium transients, which are a measure of excitation-contraction coupling, were depressed by 50% in both the inflow and outflow portions of the looped heart tube. The myocardial transients could be rescued by replacing the cardiac neural crest. The cardiac jelly produced by the myocardium was distributed in an uneven, rather than uniform, pattern. An extreme variability in external morphology could be attributed to the uneven distribution of cardiac jelly. In the absence of cardiac neural crest, the myocardium was characterized by somewhat disorganized myofibrils that may be a result of abnormally elevated proliferation. In contrast, endocardial development appeared normal, as evidenced by normal expression of fibrillin-2 protein (JB3 antigen) and normal formation of cushion mesenchyme and trabeculae. The signs of abnormal myocardial development coincident with normal endocardium suggest that the presence of cardiac neural crest cells is necessary for normal differentiation and function of the myocardium during early heart development. These results indicate a novel role for neural crest cells in myocardial maturation.

Monitoring the migrations of cells during embryonic development requires a system in which cells can be identified in situ during locomotion. One promising system involves the generation of chimeras by transplanting mouse cells into chick embryos in ovo to exploit the wealth of mouse genetic variants. The success of this technique relies on the ability to detect individual mouse cells in a chick environment with high specificity. The murine B2 family of short interspersed elements is present in the mouse genome at copy numbers in excess of 10(5), whereas this sequence is absent in the chick genome based on hybridization techniques. This differential of five orders of magnitude produces signals in mouse cells that are easily identified, even in an environment that is predominantly chick. Thus, the B2 repeat probe is highly effective for the purpose of identifying mouse cells in mouse-chick chimeras.

Ablation of the cardiac neural crest in chick embryos results in abnormal remodeling of the aortic arch arteries and absence of the cardiac outflow septum. These traditional roles of neural crest were described more than a decade ago and with the advent of molecular and transgenic techniques, we are now beginning to unravel the molecular underpinnings of these processes. Recently, it has been shown that cardiac neural crest ablation also results in defects in myocardial development that can be observed as early as stage 14, a stage at which the cardiac neural crest has not yet entered the cardiac outflow tract. These defects, which include reduced contractility, deficient excitation–contraction coupling, as determined by lowered calcium transients, and disorganized contractile apparatus, are likely due to an absence of neural crest-derived cells in the pharyngeal arch region at this stage. In normal embryos, the cardiac neural crest provides a substantial cellular partition interposed between the pharyngeal endoderm, the endothelium of the aortic arch arteries and the aortic sac and the myocardium of the outflow tract. In cardiac neural crest-ablated embryos, the geometry of these elements is disturbed such that the endoderm and endothelium are apposed leaving the myocardial cuff in much closer proximity to the pharyngeal endoderm than normal. New experimental evidence suggests that neural crest cells which normally interpose between the pharyngeal endoderm and the myocardium, suppress a signal, perhaps a growth factor, produced by the pharyngeal endoderm that deleteriously affects further myocardial development.

▪ Abstract The discovery in the chick embryo that a specific region of the neural crest, termed the cardiac neural crest, is essential for septation of the cardiac outflow tract and for aortic arch artery development has led to the classification of a whole series of human cardiac defects as neural crest-associated. Recently, several mouse genetic models have been effectively employed to yield new insights into the relationship between cardiac neural crest and structural heart development. In all the animal models of neural crest-related heart defects, prenatal mortality is too high to be attributed to structural defects of the heart alone, and there are obvious signs of severe cardiac dysfunction. The evidence indicates that poor viability is from impaired cardiac excitation-contraction coupling and contractile function at the myocyte level. The continued study of experimental and genetically defined models with neural crest-associated heart defects will prove useful in identifying the common pathways by which the neural crest contributes to normal heart development.

Ablation of cardiac neural crest results in cardiovascular malformations. Depressed ventricular contractility has been noted in cardiac neural crest-ablated embryos several hours before the time when neural crest cells would have arrived in the cardiac outflow tract and several days before the appearance of any malformations. The reason for this depressed heart function is not known. Recently, transient cranial hemorrhages were found in chick embryos during the third day of incubation, several hours before depressed ventricular contractility can be measured. We sought to determine whether depressed ventricular contractility could be associated with these transient hemorrhages. We were also interested in defining some of the factors that influence the incidence and severity of hemorrhaging. Three groups of embryos were used: cardiac neural crest-ablated, sham-operated, and unopened controls. All groups were found to experience transient hemorrhage from a common origin in the forebrain. However, the incidence and degree of hemorrhage were higher and more severe in embryos with cardiac neural crest ablation than in the other two groups. The cardiac ejection and shortening fractions were measured at stage 18 in embryos with and without hemorrhaging, and it was found that a decrease in these parameters was associated solely with ablation of cardiac neural crest and not with hemorrhage. By altering the incubation conditions, we determined that conditions that increase the oxygen in the air space are associated with increased severity and occurrence of hemorrhage and decreased viability in the first 3 days of incubation. Our results indicate that transient cranial hemorrhage does not cause depressed contractility in cardiac neural crest-ablated embryos, and increased severity of hemorrhaging is most likely due to an increase in oxygen availability. Teratology 56:300–304, 1997. © 1997 Wiley-Liss, Inc.

In a genetic mouse model of human phenylketonuria we have examined the offspring of hyperphenylalaninemic mothers for the presence of cardiovascular defects, an important feature of the pathology of the human maternal phenylketonuria syndrome. Beginning at 14.5 d after conception (75% through gestation), a variety of cardiovascular defects became apparent among the progeny of the hyperphenylalaninemic females. These defects ranged from mild to serious and correlated with the maternal but not the fetal Pah genotype. Nearly all of the defects were vascular, however, whereas the most reported in humans so far have been cardiac. The predisposing biochemical condition in this mouse disease model seems to be the same as in the human disease; elevated maternal blood phenylalanine levels concentrated across the placental barrier to produce a teratogenic developmental environment. This model for congenital cardiovascular defects should enhance two related areas of research. 1) It should allow a more thorough investigation of the relationship between maternal diet and maternal phenylketonuria birth defects, and 2) it should provide an experimental tool to gain insight into the normal process of cardiovascular development.

The preotic neural tube has a variable ability for regeneration of neural crest depending on the neuraxial level. There is robust regeneration of neural crest in the caudal midbrain/rostral hindbrain. In contrast, removal of the cardiac neural crest results in cardiovascular abnormalities suggesting the lack of regeneration in this area, although the regenerative capacity of the cardiac crest region has never been tested directly. Premigratory cardiac neural crest was ablated bilaterally using laser irradiation or extirpation by tungsten needle, and the remaining ventral neural tube was labeled with DiI to examine any neural crest regeneration from the neural tube. The results indicate that there is very little regeneration of crest cells from the cardiac region of the neural tube if the ablation is done prior to the 5-somite stage and no regeneration after the 6-somite stage with either ablation procedure. Furthermore no compensatory response occurs from the adjacent regions of the neural crest. By contrast, we were able to confirm that regeneration of neural crest occurs in the preotic rhombencephalic neural tube even after laser irradiation. An analysis in the trunk region suggests that the trunk neural tube is similar to the cardiac region in that it does not regenerate crest cells in the ventral migratory pathway after ablation. However, melanocytes generated cranial and caudal to the ablated region migrate radially and fill in the ablated region so that there is no interruption of the normal pigment pattern. This study indicates that even though there is a variable capacity for crest regeneration in the preotic neural tube, the postotic neural tube does not have such regenerative ability.

Ablation of the cardiac neural crest results in abnormal development of the aortic arch arteries leading to altered patterning of the great arteries. The cardiac outflow tract is also affected after neural crest ablation because normally a subset of neural crest cells migrates from the pharyngeal region to form the outflow septum. Using neural crest ablation, it has not been possible to separate the occurrence of aortic arch maldevelopment from cardiac outflow tract dysmorphogenesis. In order to determine whether normal aortic arch artery development is a prerequisite for normal outflow tract development, we have used a combination of antisense treatment with backtransplantation of cardiac neural folds to produce abnormal patterning of the aortic arch arteries. Paralogous groups of Hox messages with their anterior expression domains in pharyngeal arches 3, 4 and 6 were targeted. Antisense targeted to paralogous group 3 Hox message caused aortic arch 3 located within the pharyngeal arch to regress in a manner similar to aortic arch 2, while antisense targeted to paralogous group 5 Hox message caused the appearance of an additional pharyngeal arch containing a novel and completely independent aortic arch artery. Antisense treatment targeting paralogous group 4 Hox message led to no detectable cardiovascular phenotype in the first 6 days of development. While regression of arch artery 3 was associated with abnormal branching patterns of the aorta and pulmonary trunk, this did not involve abnormal separation of the aorta and pulmonary trunks, the semilunar valves or the subvalvular region of the outflow tract. Because none of these changes in pharyngeal or aortic arch artery development was accompanied by abnormal development of the cardiac outflow tract, it appears that normal patterning of the aortic arch arteries is not a prerequisite for normal heart development. Using reverse transcription polymerase chain reaction (RT-PCR) we were unable to detect changes in any of the Hox messages except group 4, thus, using this particular experimental strategy, we are unable to demonstrate or refute that expression of hox genes by cardiac neural crest cells controls aortic arch patterning. Development of the cardiac outflow tract was normal in each instance. This suggests that abnormal aortic arch patterning does not necessarily lead to cardiac malformations. Dev Dyn 208: 34–47, 1997. © 1997 Wiley-Liss, Inc.

The neural crest constitutes a developmental field which is a morphogenetically reactive unit of the embryo. Disruption of this developmental field causes a constellation of anomalies to occur. Clustering of phenotypic abnormalities has allowed clinicians to recognize neural crest-associated syndromes with developmental abnormalities of the cardiovascular system, head, and neck. Basic research is beginning to unravel how these phenotypic characteristics are related to specific gene defects expressed during development. Currently, we do not know a one-to-one relationship between phenotypes and genotypes. These neonates with neural crest-associated conotruncal defects are born with types and genotypes. These neonates with neural crest-associated conotruncal defects are born with recognizable complex cyanotic heart defects that are ductal-dependent. It may be difficult to judge if they have DiGeorge or velocardiofacial syndromes; thus, genetic counseling is of importance. Besides their life-threatening cardiovascular defects, these neonates frequently have either transient or persistent hypocalcemia or severe immunodeficiencies that require critical care management. This review will focus on the basic research underpinnings and currently recommended clinical care of infants with neural crest-associated conotruncal defects.

Abstract During fetal development, a specialized vessel, the ductus arteriosus, shunts blood from the pulmonary artery to the aorta, thus bypassing the lungs. The ductus differs primarily from the great vessels in that it is a muscular rather than an elastic artery, and the etiology of this differential development remains controversial. We present evidence that retinoic acid (RA) may contribute to the unique muscle phenotype of the ductus arteriosus. Using a transgenic mouse carrying an RA response element– lacZ transgene that expresses β-galactosidase (β-gal) in response to endogenous RA signals during embryonic and fetal development, we observe a strong β-gal signal in the ductus arteriosus. By immunofluorescence, this signal colocalizes with the expression of the adult-specific smooth muscle myosin heavy chain isoform, SM2. The β-gal signal is present throughout fetal development and persists in the neonate until the ductus arteriosus is completely closed. β-Gal–positive cells are first detected by immunofluorescence at 13.5 days postcoitum (dpc) in the mesenchyme surrounding the ductus. By 15.5 dpc, very intense β-gal staining localizes to the ductus arteriosus but is absent or minimal in the pulmonary trunk and aortic arch; by 17.5 dpc, the smooth muscle layers of the tunica media in the ductus arteriosus exhibit positive β-gal staining. Immunostaining with antibodies against smooth muscle myosins shows that, while SM1 is expressed in all embryonic vessels, SM2 is precociously expressed in the ductus arteriosus. Furthermore, SM2 expression can be detected in the ductus as early as 15.5 dpc. In the neonate, the β-gal signal persists in the smooth muscle layer of the ductus and immunostaining colocalizes with SM2 expression. These data suggest that RA may play a role in inducing and maintaining smooth muscle differentiation in the developing ductus arteriosus and may promote precocious expression of the adult vascular phenotype.

Ablation of premigratory cardiac neural crest has been used to produce and study extensively a model of abnormal cardiovascular dysmorphology. Previous and continuing research in this laboratory concerns different aspects of the involvement of cranial neural crest in the development of cranial, cervical and cardiac tissues in chick embryos. Recently, we detected the occurrence of transient cranial hemorrhages 24-48 hr after the ablation of selected segments of premigratory cranial neural crest. Since one possible mechanism of action for certain teratogens involves nonreparable damage to a primordial embryonic tissue by an antecedent hemorrhage, the objective of this study was to determine which of three different neural crest ablations is associated with hemorrhages and whether subsequent congenital abnormalities were correlated with the ablation procedure and/or hemorrhage. Premigratory neural crest was ablated from 3 different sites, designated cardiac, mesencephalic and trunk crest, respectively, of stage 8-10 chick embryos. Sham-operated embryos were controls. At 24, 30, and 48 hr after ablation, each embryo was observed for the presence of hemorrhages. On incubation day 11 all the living embryos were killed, fixed, weighed, and analyzed for selected length measurements, developmental stage, and the types and rates of congenital abnormalities. Cardiac and mesencephalic ablation group embryos exhibited significant incidences of cranial hemorrhages and changes in many of the parameters analyzed. It was concluded that the cardiac and mesencephalic, but not the trunk neural crest ablations, produced significant changes in incubation day 11 embryos. However, there was no correlation between the abnormalities and the prior occurrence of the transient cranial hemorrhages.

Double-label immunohistochemistry was used to compare early aortic arch artery development in cardiac neural crest-ablated and sham-operated quail embryos ranging from stage 13 to stage 18. The monoclonal antibody QH-1 labeled endothelial cells and their precursors, and HNK-1 labeled migrating neural crest cells. In the sham-operated embryos, the third aortic arch artery developed from a lumenizing strand of endothelial precursors that became separated from the pharyngeal endoderm by migrating cardiac neural crest cells as they ensheathed the artery. The arch artery of the neural crest-ablated embryos lumenized but failed to become separated from the pharyngeal endoderm, indicating that neural crest is unnecessary for the early formation of the aortic arch artery. However, once blood flow was initiated through the third arch artery of crest-ablated embryos at stage 16, the artery became misshapen and sinusoidal. By embryonic day 3, abnormal connections to the dorsal aorta occurred and bilateral symmetry was lost, suggesting that the loss of neural crest-derived ectomesenchyme destabilizes the nascent artery. Although here we show no loss of the third arch artery, past studies have reported hypoplasia or missing carotids in older neural crest-ablated embryos (Bockman et al. [1987] Am. J. Anat. 180:332–341; Bockman et al. [1989] Anat. Rec. 225:209–217; Nishibatake et al. [1987] Circulation 75:255–264; Tomita et al. [1991] Circulation 84:1289–1295). We suggest that the cardiac neural crest is essential for the persistence of an arch artery, but not its formation. Furthermore, since changes in the development of the arch artery are seen prior to the formation of the tunica media, it is suggested that a critical period is reached in the development of the arch artery, after lumenization, but prior to the formation of the tunica media, which necessitates the presence of the cardiac neural crest. © 1996 Wiley-Liss, Inc.

The int-2 gene was first described as a common proviral integration site in tumors induced by mouse mammary tumor virus (MMTV). During embryonic development int-2 is produced and released by cells in the rhombencephalon and diffuses to the ectoderm to induce formation of the otocyst from the otic placode. Int-2 also influences the development of the vestibulo-cochlear ganglion that is derived from the otic placode. During embryogenesis the otic and nodose placodes, primordia of the inner ear and the nodose ganglia, respectively, are located adjacent to each other in the embryonic ectoderm. The nodose ganglia provide sensory innervation to all of the viscera. Using Northern analysis we determined that a high level of int-2 is transcribed in stage 14 chick embryos. This is the time when cells begin to migrate from the nodose placodes to form the nodose ganglia. Using human and mouse sequences to design primers around the translation start site of the transcript, a partial clone containing the translation start site of chick int-2 was obtained by polymerase chain reaction amplification from chick genomic DNA and cloned. An antisense oligodeoxynucleotide was designed to the region of the translation start site, and in vitro and in vivo techniques were used to demonstrate that inhibition of int-2 translation using this antisense oligonucleotide causes delayed and abnormal development of the nodose placodes. For in vitro studies, explants of stage 12 chick embryos containing neural tube, adjacent surface ectoderm, and pharyngeal endoderm were cultured with int-2 antisense oligonucleotide. For the in vivo studies, pieces of resin impregnated with int-2 antisense oligonucleotide were implanted into the neural tube of stage 12 chick embryos at the level of the otic and nodose placodes. We found that the development of the nodose placodes exposed to int-2 antisense oligodeoxynucleotide was delayed and abnormal. These differences were not seen in embryos or explants treated with similar concentrations of sense or nonspecific oligomers. Western analysis and immunohistochemistry showed that an int-2-immunoreactive protein was reduced in the pharyngeal region and nodose ganglia of the embryos exposed to int-2 antisense oligodeoxynucleotide, whereas it was not affected in embryos treated with sense oligomer. We conclude that int-2 may be necessary for normal development of the nodose ganglia.

Using the conserved basic helix-loop-helix (bHLH) domain of AS-C and MASH, we amplified by polymerase chain reaction, the HLH region of a chick AS-C/MASH homologue from cDNA prepared from poly (A+) RNA isolated from a region rich in cardiac neural crest cells. A full-length cDNA clone was obtained from a stage 23 chick head cDNA library using this conserved sequence as a probe. Comparison of this clone with MASH1 revealed strong homologies in the bHLH domain and carboxyl terminal region of the conceptually translated protein. The clone was named chick achaete-scute homologue (CASH). This transcript has a single open reading frame that codes for a protein of 218 residues. The deduced amino acid sequence of CASH is 100% identical to MASH1 in the bHLH region and 83.9% identical over the entire protein. The bHLH domain of CASH is highly conserved within the AS-C family. Northern analysis indicates that CASH is expressed transiently in a defined spatial pattern. The highest expression is in the head between stages 23 and 26. Whole mount in situ hybridization suggests that CASH is expressed at stage 23 in the dorsal ventricular zone and central sensory pathways and later in the ventral ventricular zone and ventral horn. By stage 28, it is expressed ubiquitously in the peripheral nervous system including all of the neurons derived from the the neural crest and epipharyngeal placodes, as well as neural crest-derived chromaffin cells in the adrenal medulla. Amplification by polymerase chain reaction indicates that CASH is expressed in the cardiac neural fold and continues to be expressed in cultured cardiac neural crest cells.

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 nodose 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.

Ablation of the cardiac neural crest has provided a powerful model of cardiovascular dysmorphogenesis because removal or manipulation of neural crest cells can be done before migration in sites distant from central cardiovascular development in chick embryos. Because the heart begins to function so early in its own morphogenetic history, the cardiovascular system is exquisitely sensitive to the embryonic environment, and virtually any manipulation of the embryo has cardiovascular consequences. Thus, although neural crest manipulation is not devoid of experimental artifact, it has resulted in significant advances in our understanding of certain of the factors involved in normal heart development in a way that direct manipulation of heart development does not. We have now identified two major roles for the neural crest in cardiovascular patterning. These are (1) participation in the patterning of the pharyngeal arches and their derivatives, including the aortic arch arteries, which will become the great arteries of the thorax, and (2) migration of a discrete population of neural crest cells into the cardiac outflow tract and participation in formation of the outflow septum. With the advent of genetically based animal models of cardiovascular dysmorphogenesis, some of which are neural crest related, it becomes important to understand what we know definitively from the ablation model along with the questions that remain. This review will focus on the roles of the neural crest in cardiovascular patterning as seen in the ablation model. An important part of the emerging neural crest–ablation phenotype is an early change in the functional competency of the developing myocardium. We believe that the neural crest–ablation phenotype provides a prototype for comparison with newer models of neural crest–related cardiovascular dysmorphogenesis and that the changes in myocardial functional development provide a means of understanding the early mortality associated with neural crest–type cardiac dysmorphogenesis. The four-chambered heart …

Neural crest cells originating from the posterior rhombencephalon migrate to the cardiac outflow tract and participate in division of the aorta and pulmonary trunk. Ablation of this region of premigratory neural crest in the chick embryos results in nondivision of the outflow vessels. Subtractive hybridization was used to identify messages in the outflow tract of embryos with cardiac neural crest cells versus those lacking the cardiac neural crest cells. The chick L10 ribosomal protein was found to be differentially expressed in the outflow tract of embryos with cardiac neural crest. Several conditions have been identified that involve differential expression of ribosomal proteins, but this is the first report of differential expression in eukaryotic embryonic development.

The phenotype of somatic cells has recently been found to be reversible. Direct reprogramming of one cell type into another has been achieved with transduction and over expression of exogenous defined transcription factors emphasizing their role in specifying cell fate. To discover early and novel endogenous transcription factors that may have a role in adult-derived stem cell acquisition of a cardiomyocyte phenotype, mesenchymal stem cells from human and mouse bone marrow and rat liver were co-cultured with neonatal cardiomyocytes as an in vitro cardiogenic microenvironment. Cell-cell communications develop between the two cell types as early as 24 hrs in co-culture and are required for elaboration of a myocardial phenotype in the stem cells 8-16 days later. These intercellular communications are associated with novel Ca(2+) oscillations in the stem cells that are synchronous with the Ca(2+) transients in adjacent cardiomyocytes and are detected in the stem cells as early as 24-48 hrs in co-culture. Early and significant up-regulation of Ca(2+)-dependent effectors, CAMTA1 and RCAN1 ensues before a myocardial program is activated. CAMTA1 loss-of-function minimizes the activation of the cardiac gene program in the stem cells. While the expression of RCAN1 suggests involvement of the well-characterized calcineurin-NFAT pathway as a response to a Ca(2+) signal, the CAMTA1 up-regulated expression as a response to such a signal in the stem cells was unknown. Cell-cell communications between the stem cells and adjacent cardiomyocytes induce Ca(2+) signals that activate a myocardial gene program in the stem cells via a novel and early Ca(2+)-dependent intermediate, up-regulation of CAMTA1.

Defective development of the cardiac outflow tract results in congenital cardiac anomalies that are known clinically as conotruncal defects. Development of the outflow tract is complicated, and the large number of conotruncal malformations that are seen in children reflects this complexity. Historically, knowledge of the embryogenesis of any heart defects has been difficult to attain because of the lack of models in which the pathogenesis of the defect could be studied prospectively. It is not difficult to induce cardiac malformations experimentally in animals but such malformations are not consistently of the same type, even using the same experimental paradigm. It is difficult to study a particular outflow malformation in a model with only 20% or even 50% incidence of the malformation. There is another problem with outflow development that further confounds our understanding: many but not all of the outflow malformations are related to each other in that they represent more or less severe manifestations of the same developmental pathology.1 The retinoic acid model was one of the earliest teratogenic models of heart defects in the 1940s and 50s.2 A strain of keeshound dog developed by Patterson became the first genetic model to be studied extensively.3 Both of these models provided information about the morphology of some defects at various stages of development, but these models said little about the pathogenesis of the defects because of the problems mentioned above, ie, variability and low penetrance of any particular phenotype. The first and (possibly) most informative model of heart defects was the neural crest ablation model in chick embryos.4 This model produced an incidence of 90% persistent truncus arteriosus and has become the “gold standard” for this type of defect. Although this model led to a number of misunderstandings about the defects that can be directly attributed …

The secondary heart field is a conserved developmental domain in avian and mammalian embryos that contributes myocardium and smooth muscle to the definitive cardiac arterial pole. This field is part of the overall heart field and its myocardial component has been fate mapped from the epiblast to the heart in both mammals and birds. In this study we show that the population that gives rise to the arterial pole of the zebrafish can be traced from the epiblast, is a discrete part of the mesodermal heart field, and contributes myocardium after initial heart tube formation, giving rise to both smooth muscle and myocardium. We also show that Isl1, a transcription factor associated with undifferentiated cells in the secondary heart field in other species, is active in this field. Furthermore, Bmp signaling promotes myocardial differentiation from the arterial pole progenitor population, whereas inhibiting Smad1/5/8 phosphorylation leads to reduced myocardial differentiation with subsequent increased smooth muscle differentiation. Molecular pathways required for secondary heart field development are conserved in teleosts, as we demonstrate that the transcription factor Tbx1 and the Sonic hedgehog pathway are necessary for normal development of the zebrafish arterial pole.

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.

The cardiac neural crest is essential for normal development of the cardiovascular system. Cardiac neural crest cells are derived from the neural folds located between the mid-otic placodes and the caudal limit of somite 3. These crest cells can differentiate into a variety of mesenchymal cell types that support cardiovascular development, in addition to neurogenic cells. When cultured, many expressα-smooth muscle actin or neurofilaments and lose their undifferentiated neural crest phonotype as shown by a decrease in HNK-1 reactivity. We wanted to determine whether cultured cardiac neural crest cells maintained the potency to support normal heart development when backtransplanted into embryos lacking their native cardiac neural crest. Under usual circumstances removal of the cardiac neural crest results in 80–100% incidence of persistent truncus arteriosus. The present study report a system in which cardiac neural folds are cultured for 3 days and the cells backtransplanted into chick embryos after laser-induced ablation of the intrinsic cardiac neural folds. Rescue of heart development was improved 50% when cultured cells were backtransplanted and almost 200% when the backtransplanted cells had been cultured in leukemia inhibitory factor (LIF). To determine whether the cultured cells are capable of following normal migratory routes, cultured homospecific cardiac neural crest cells were tagged with DiI. Initially, fluorescent cells were found concentrated around the neural tube. By the second day following backtransplantation, the cells had migrated to the circumpharyngeal crest, populated the pharyngeal arches and aortic arch arteries, and were in the region of the cardiac outflow tract. By the third day, the labeled cells had dispersed, but could be found around the neural tube, esophagus, cardiac outflow tract, and within the dorsal root ganglia. Interestingly, a cranial migration to the periphery of the eyes was also noted. With the exception of the cranial migration to the eyes, cultured and backtransplanted cardiac neural crest cells followed normal migratory pathways to the cardiac outflow tract. LIF is used for the in vitro maintenance of the pluripotential phenotype of embryonic stem cells. In an effort to understand why LIF improves the ability of cultured neural crest cells to support normal heart development, we have examined the relationship of neural crest expression of HNK-1 antigen,α-smooth muscle actin, and neurofilament protein in neural crest cells cultured in LIF. LIF treatment resulted in an expanded period of expression of HNK-1 antigen, associated with a decrease in expression ofα-smooth muscle actin. The expanded period of HNK-1 expression is associated with proliferation of the HNK-1 positive cell population. Expression of the neurofilament was the same in cells cultured with or without LIF. © 1993 Wiley-Liss, Inc.

Previous studies of cardiac neural crest (CNC) migration in early chick embryos demonstrated CNC cells in the media of pharyngeal arch arteries three, four, and six, and in the most proximal part of the developing pulmonary arteries. The objectives of this study were to learn 1) to what extent the CNC is involved in the later development of the pulmonary arteries, 2) how the CNC cells are distributed in the sixth aortic arch artery including the wall of the ductus arteriosus in the older embryo, and 3) what happens to the CNC as the pulmonary artery/sixth arch complex grows into its adult configuration. Quail-to-chick chimeras were used to study CNC distribution in embryos aged 6 to 18 days. Controls (undisturbed chick embryos) were collected with chimeras. Each was fixed, processed, sectioned, stained with Feulgen-Rossenbeck stain, and analyzed. The results demonstrated that CNC disappeared from the proximal pulmonary arteries by embryonic day 9 and played no further role in pulmonary artery development. With the exception of the endothelium, CNC completely filled the wall of the sixth aortic arch artery as far distally as its junction with the dorsal aorta in younger embryos and with the aorta in older embryos, thus suggesting the possibility of proximodistal migration of CNC along the sixth aortic arch. The ductus wall, filled with CNC, was intimately associated with the recurrent laryngeal nerve, also filled with CNC, thereby strongly suggesting a role for CNC in ductal closure.

The vagal neural crest adjacent to the first seven somites gives rise to both ganglionic and ectomesenchymal derivatives. Ganglionic derivatives are the neurons and supportive cells of the enteric nervous system (ENS), cardiac, and dorsal root ganglia. Ectomesenchymal derivatives are cells in the cardiac outflow tract and the mesenchymal components of thymus and parathyroids. Ectomesenchymal derivatives are formed by a segment of the vagal neural crest, from the level of the otic vesicle down to the caudal boundary of the third somite, called the cardiac neural crest. We performed neural crest ablations to study regional differences within the avian vagal neural crest with regard to the formation of the ENS. Ablation of the entire vagal neural crest from the mid-otic vesicle down to the seventh somite plus the nodose placode resulted in the absence of ganglia in the midgut (jejunum and ileum) and hindgut (colon). The foregut (esophagus, proventriculus, gizzard, and duodenum) was normally innervated. After ablation of the vagal neural crest adjacent to somites 3-5, ganglia were absent in the hindgut. Ablations of vagal neural crest not including this segment had no effect on the formation of the ENS. We surmise that the innervation of the hindgut in vivo depends specifically on the neural crest adjacent to somites 3-5, whereas innervation of the midgut can be accomplished by all segments within the vagal neural crest. The foregut can also be innervated by a source outside the vagal neural crest. To study intrinsic differences between various vagal neural crest segments regarding ENS formation, we performed chorioallantoic membrane cocultures of segments of quail vagal neural anlage and E4 chicken hindgut. We found that all vagal neural crest segments were able to give rise to enteric ganglia in the hindgut. When the neural crest of somites 6 and 7 was included in the segment, we also found melanocytes in the hindgut, suggesting that this segment is more related to trunk neural crest. Furthermore, we found that the vagal neural anlage from older embryos (> 18 somites) showed an increased potential to form enteric ganglia. This suggests that vagal neural crest cells that have been in prolonged contact with the neural tube in vivo, because of either late emigration or delayed migration, have an increased probability to form enteric ganglia.