Temperature-activated ion channels in neural crest cells confer maternal fever-associated birth defects.

Birth defects of the heart and face are common, and most have no known genetic cause, suggesting a role for environmental factors. Maternal fever during the first trimester is an environmental risk factor linked to these defects. Neural crest cells are precursor populations essential to the development of both at-risk tissues. We report that two heat-activated transient receptor potential (TRP) ion channels, TRPV1 and TRPV4, were present in neural crest cells during critical windows of heart and face development. TRPV1 antagonists protected against the development of hyperthermia-induced defects in chick embryos. Treatment with chemical agonists of TRPV1 or TRPV4 replicated hyperthermia-induced birth defects in chick and zebrafish embryos. To test whether transient TRPV channel permeability in neural crest cells was sufficient to induce these defects, we engineered iron-binding modifications to TRPV1 and TRPV4 that enabled remote and noninvasive activation of these channels in specific cellular locations and at specific developmental times in chick embryos with radio-frequency electromagnetic fields. Transient stimulation of radio frequency-controlled TRP channels in neural crest cells replicated fever-associated defects in developing chick embryos. Our data provide a previously undescribed mechanism for congenital defects, whereby hyperthermia activates ion channels that negatively affect fetal development.

Loss of Wnt5a disrupts second heart field cell deployment and may contribute to OFT malformations in DiGeorge syndrome.

Outflow tract (OFT) malformation accounts for ∼30% of human congenital heart defects and manifests frequently in TBX1 haplo-insufficiency associated DiGeorge (22q11.2 deletion) syndrome. OFT myocardium originates from second heart field (SHF) progenitors in the pharyngeal and splanchnic mesoderm (SpM), but how these progenitors are deployed to the OFT is unclear. We find that SHF progenitors in the SpM gradually gain epithelial character and are deployed to the OFT as a cohesive sheet. Wnt5a, a non-canonical Wnt, is expressed specifically in the caudal SpM and may regulate oriented cell intercalation to incorporate SHF progenitors into an epithelial-like sheet, thereby generating the pushing force to deploy SHF cells rostrally into the OFT. Using enhancer trap and Cre transgenes, our lineage tracing experiments show that in Wnt5a null mice, SHF progenitors are trapped in the SpM and fail to be deployed to the OFT efficiently, resulting in a reduction in the inferior OFT myocardial wall and its derivative, subpulmonary myocardium. Concomitantly, the superior OFT and subaortic myocardium are expanded. Finally, in chick embryos, blocking the Wnt5a function in the caudal SpM perturbs polarized elongation of SHF progenitors, and compromises their deployment to the OFT. Collectively, our results highlight a critical role for Wnt5a in deploying SHF progenitors from the SpM to the OFT. Given that Wnt5a is a putative transcriptional target of Tbx1, and the similar reduction of subpulmonary myocardium in Tbx1 mutant mice, our results suggest that perturbing Wnt5a-mediated SHF deployment may be an important pathogenic mechanism contributing to OFT malformations in DiGeorge syndrome.

Calcium signaling regulates ventricular hypertrophy during development independent of contraction or blood flow.

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 Ca(2+)-dependent growth pathways that function independent of hemodynamic forces. To test this hypothesis, zebrafish embryos were treated with inhibitors or activators of Ca(2+) signaling in the presence or absence of contraction during the period of chamber development. Abolishment of contractile function alone in the setting of preserved Ca(2+) signaling did not impair ventricular hypertrophy. In contrast, inhibition of L-type voltage-gated Ca(2+) influx abolished contraction and led to reduced ventricular hypertrophy, whereas increasing L-type voltage-gated Ca(2+) influx led to enhanced ventricular hypertrophy in either the presence or absence of contraction. Similarly, inhibition of the downstream Ca(2+)-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 Ca(2+) influx and contraction by expression of a constitutively active calcineurin. These data suggest that ventricular cardiomyocyte hypertrophy during chamber formation is dependent upon Ca(2+) signaling pathways that are unaffected by heart function or hemodynamic forces. Disruption of Ca(2+)-dependent hypertrophy during heart development may therefore represent one mechanism for impaired chamber formation that is not related to impaired blood flow.

Evolutionary and developmental origins of the cardiac neural crest: building a divided outflow tract.

The cardiac neural crest cells (CNCCs) have played an important role in the evolution and development of the vertebrate cardiovascular system: from reinforcement of the developing aortic arch arteries early in vertebrate evolution, to later orchestration of aortic arch artery remodeling into the great arteries of the heart, and finally outflow tract septation in amniotes. A critical element necessary for the evolutionary advent of outflow tract septation was the co-evolution of the cardiac neural crest cells with the second heart field. This review highlights the major transitions in vertebrate circulatory evolution, explores the evolutionary developmental origins of the CNCCs from the third stream cranial neural crest, and explores candidate signaling pathways in CNCC and outflow tract evolution drawn from our knowledge of DiGeorge Syndrome.

Follistatin in chondrocytes: the link between TRPV4 channelopathies and skeletal malformations.

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 TRPV4(V620I) 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 the TRPV4(V620I) 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 TRPV4(T89I) mutation. Taken together, these data strongly support that up-regulation of FST in chondrocytes by skeletal dysplasia-inducing TRPV4 mutations contributes to disease pathogenesis.

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.

Factors controlling cardiac neural crest cell migration.

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.

BMP signaling modulates hedgehog-induced secondary heart field proliferation.

Sonic hedgehog signaling in the secondary heart field has a clear role in cardiac arterial pole development. In the absence of hedgehog signaling, proliferation is reduced in secondary heart field progenitors, and embryos predominantly develop pulmonary atresia. While it is expected that proliferation in the secondary heart field would be increased with elevated hedgehog signaling, this idea has never been tested. We hypothesized that up-regulating hedgehog signaling would increase secondary heart field proliferation, which would lead to arterial pole defects. In culture, secondary heart field explants proliferated up to 6-fold more in response to the hedgehog signaling agonist SAG, while myocardial differentiation and migration were unaffected. Treatment of chick embryos with SAG at HH14, just before the peak in secondary heart field proliferation, resulted unexpectedly in stenosis of both the aortic and pulmonary outlets. We examined proliferation in the secondary heart field and found that SAG-treated embryos exhibited a much milder increase in proliferation than was indicated by the in vitro experiments. To determine the source of other signaling factors that could modulate increased hedgehog signaling, we co-cultured secondary heart field explants with isolated pharyngeal endoderm or outflow tract and found that outflow tract co-cultures prevented SAG-induced proliferation. BMP2 is made and secreted by the outflow tract myocardium. To determine whether BMP signaling could prevent SAG-induced proliferation, we treated explants with SAG and BMP2 and found that BMP2 inhibited SAG-induced proliferation. In vivo, SAG-treated embryos showed up-regulated BMP2 expression and signaling. Together, these results indicate that BMP signaling from the outflow tract modulates hedgehog-induced proliferation in the secondary heart field.

A caudal proliferating growth center contributes to both poles of the forming heart tube.

Recent studies have shown that the primary heart tube continues to grow by addition of cells from the coelomic wall. This growth occurs concomitantly with embryonic folding and formation of the coelomic cavity, making early heart formation morphologically complex. A scarcity of data on localized growth parameters further hampers the understanding of cardiac growth. Therefore, we investigated local proliferation during early heart formation. Firstly, we determined the cell cycle length of primary myocardium of the early heart tube to be 5.5 days, showing that this myocardium is nonproliferating and implying that initial heart formation occurs solely by addition of cells. In line with this, we show that the heart tube rapidly lengthens at its inflow by differentiation of recently divided precursor cells. To track the origin of these cells, we made quantitative 3D reconstructions of proliferation in the forming heart tube and the mesoderm of its flanking coelomic walls. These reconstructions show a single, albeit bilateral, center of rapid proliferation in the caudomedial pericardial back wall. This center expresses Islet1. Cell tracing showed that cells from this caudal growth center, besides feeding into the venous pole of the heart, also move cranially via the dorsal pericardial mesoderm and differentiate into myocardium at the arterial pole. Inhibition of caudal proliferation impairs the formation of both the atria and the right ventricle. These data show how a proliferating growth center in the caudal coelomic wall elongates the heart tube at both its venous and arterial pole, providing a morphological mechanism for early heart formation.

Model systems for the study of heart development and disease. Cardiac neural crest and conotruncal malformations.

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.

Cardiac arterial pole alignment is sensitive to FGF8 signaling in the pharynx.

Morphogenesis of the cardiac arterial pole is dependent on addition of myocardium and smooth muscle from the secondary heart field and septation by cardiac neural crest cells. Cardiac neural crest ablation results in persistent truncus arteriosus and failure of addition of myocardium from the secondary heart field leading to malalignment of the arterial pole with the ventricles. Previously, we have shown that elevated FGF signaling after neural crest ablation causes depressed Ca2+ transients in the primary heart tube. We hypothesized that neural crest ablation results in elevated FGF8 signaling in the caudal pharynx that disrupts secondary heart field development. In this study, we show that FGF8 signaling is elevated in the caudal pharynx after cardiac neural crest ablation. In addition, treatment of cardiac neural crest-ablated embryos with FGF8b blocking antibody or an FGF receptor blocker rescues secondary heart field myocardial development in a time- and dose-dependent manner. Interestingly, reduction of FGF8 signaling in normal embryos disrupts myocardial secondary heart field development, resulting in arterial pole malalignment. These results indicate that the secondary heart field myocardium is particularly sensitive to FGF8 signaling for normal conotruncal development, and further, that cardiac neural crest cells modulate FGF8 signaling in the caudal pharynx.

Cardiac neural crest is necessary for normal addition of the myocardium to the arterial pole from the secondary heart field.

In cardiac neural-crest-ablated embryos, the secondary heart field fails to add myocardial cells to the outflow tract and elongation of the tube is deficient. Since that study, we have shown that the secondary heart field provides both myocardium and smooth muscle to the arterial pole. The present study was undertaken to determine whether addition of both cell types is disrupted after neural crest ablation. Marking experiments confirm that the myocardial component fails to be added to the outflow tract after neural crest ablation. The cells destined to go into the outflow myocardium fail to migrate and are left at the junction of the outflow myocardium with the nascent smooth muscle at the base of the arterial pole. In contrast, the vascular smooth muscle component is added to the arterial pole normally after neural crest ablation. When the myocardium is not added to the outflow tract, the point where the outflow joins the pharynx does not move caudally as it normally should, the aortic sac is smaller and fails to elongate resulting in abnormal connections of the outflow tract with the caudal aortic arch arteries.

Secondary heart field contributes myocardium and smooth muscle to the arterial pole of the developing heart.

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.

Expression of ZIC genes in the development of the chick inner ear and nervous system.

ZIC genes, vertebrate homologues of the Drosophila pair-rule gene odd-paired (opa), function in embryonic pattern formation, in the early stages of central nervous system neurogenesis and in cerebellar maturation. Mouse Zic genes are expressed in restricted, and in some cases overlapping, patterns during development, particularly in the central and peripheral nervous systems. We identified chick ZIC2 in a differential display analysis of the auditory system designed to find genes up-regulated after noise trauma. In this study, we examined the expression of chick ZIC1, ZIC2, and ZIC3 by in situ hybridization in normal inner ear development and in the tissues that influence its development, including the hindbrain, the neural crest, and the periotic mesenchyme. Between Hamburger and Hamilton stages 13 and 24, all three ZIC genes were found in the dorsal periotic mesenchyme adjacent to the developing inner ear. ZIC1 mRNA was expressed in the otocyst epithelium between stages 12 and 24, in some sensory tissue, as well as in a striped pattern in the floorplate of the hindbrain that appears to be complementary to that of Chordin, a gene known to regulate ZIC expression in frogs. Chick ZIC genes are also expressed in the neuroectoderm, paraxial mesenchyme, brain, spinal cord, neural crest, and/or the overlying ectoderm as well as the limb buds. In general, ZIC1 and ZIC2 expression patterns overlapped, although ZIC2 expression was less robust; ZIC3 expression was minimal. These observations suggest that ZIC genes, in addition to their known roles in brain development, may play an important role in the development of the chick inner ear.