Cardiac neural crest ablation alters Id2 gene expression in the developing heart

Id proteins are negative regulators of basic helix-loop-helix gene products and participate in many developmental processes. We have evaluated the expression of Id2 in the developing chick heart and found expression in the cardiac neural crest, secondary heart field, outflow tract, inflow tract, and anterior parasympathetic plexus. Cardiac neural crest ablation in the chick embryo, which causes structural defects of the cardiac outflow tract, results in a significant loss of Id2 expression in the outflow tract. Id2 is also expressed in Xenopus neural folds, branchial arches, cardiac outflow tract, inflow tract, and splanchnic mesoderm. Ablation of the premigratory neural crest in Xenopus embryos results in abnormal formation of the heart and a loss of Id2 expression in the heart and splanchnic mesoderm. This data suggests that the presence of neural crest is required for normal Id2 expression in both chick and Xenopus heart development and provides evidence that neural crest is involved in heart development in Xenopus embryos.     

Heparan Sulfate Biosynthesis Enzyme,Ext1, Contributes to Outflow Tract Development of Mouse Heart via Modulation of FGF Signaling

Glycosaminoglycans are important regulators of multiple signaling pathways. As a major constituent of the heart extracellular matrix, glycosaminoglycans are implicated in cardiac morphogenesis through interactions with different signaling morphogens. Ext1 is a glycosyltransferase responsible for heparan sulfate synthesis. Here, we evaluate the function of Ext1 in heart development by analyzing Ext1 hypomorphic mutant and conditional knockout mice. Outflow tract alignment is sensitive to the dosage of Ext1. Deletion of Ext1 in the mesoderm induces a cardiac phenotype similar to that of a mutant with conditional deletion of UDP-glucose dehydrogenase, a key enzyme responsible for synthesis of all glycosaminoglycans. The outflow tract defect in conditional Ext1 knockout(Ext1 ^f/f:Mesp1Cre) mice is attributable to the reduced contribution of second heart field and neural crest cells. Ext1 deletion leads to downregulation of FGF signaling in the pharyngeal mesoderm. Exogenous FGF8 ameliorates the defects in the outflow tract and pharyngeal explants. In addition, Ext1 expression in second heart field and neural crest cells is required for outflow tract remodeling. Our results collectively indicate that Ext1 is crucial for outflow tract formation in distinct progenitor cells, and heparan sulfate modulates FGF signaling during early heart development.     

Cardiac neural crest is dispensable for outflow tract septation in Xenopus

In vertebrate embryos, cardiac precursor cells of the primary heart field are specified in the lateral mesoderm. These cells converge at the ventral midline to form the linear heart tube, and give rise to the atria and the left ventricle. The right ventricle and the outflow tract are derived from an adjacent population of precursors known as the second heart field. In addition, the cardiac neural crest contributes cells to the septum of the outflow tract to separate the systemic and the pulmonary circulations. The amphibian heart has a single ventricle and an outflow tract with an incomplete spiral septum; however, it is unknown whether the cardiac neural crest is also involved in outflow tract septation, as in amniotes. Using a combination of tissue transplantations and molecular analyses in Xenopus we show that the amphibian outflow tract is derived from a second heart field equivalent to that described in birds and mammals. However, in contrast to what we see in amniotes, it is the second heart field and not the cardiac neural crest that forms the septum of the amphibian outflow tract. In Xenopus, cardiac neural crest cells remain confined to the aortic sac and arch arteries and never populate the outflow tract cushions. This significant difference suggests that cardiac neural crest cell migration into the cardiac cushions is an amniote-specific characteristic, presumably acquired to increase the mass of the outflow tract septum with the evolutionary need for a fully divided circulation.     

Type 1 Fibroblast Growth Factor Receptor in Cranial Neural Crest Cell-derived Mesenchyme Is Required for Palatogenesis

Cleft palate is a common congenital birth defect. The fibroblast growth factor (FGF) family has been shown to be important for palatogenesis, which elicits the regulatory functions by activating the FGF receptor tyrosine kinase. Mutations in Fgf or Fgfr are associated with cleft palate. To date, most mechanistic studies on FGF signaling in palate development have focused on FGFR2 in the epithelium. Although Fgfr1 is expressed in the cranial neural crest (CNC)-derived palate mesenchyme and Fgfr1 mutations are associated with palate defects, how FGFR1 in palate mesenchyme regulates palatogenesis is not well understood. Here, we reported that by using Wnt1^Cre to delete Fgfr1 in neural crest cells led to cleft palate, cleft lip, and other severe craniofacial defects. Detailed analyses revealed that loss-of-function mutations in Fgfr1 did not abrogate patterning of CNC cells in palate shelves. However, it upset cell signaling in the frontofacial areas, delayed cell proliferation in both epithelial and mesenchymal compartments, prevented palate shelf elevation, and compromised palate shelf fusion. This is the first report revealing how FGF signaling in CNC cells regulates palatogenesis.     

The fibroblast growth factor signaling axis controls cardiac stem cell differentiation through regulating autophagy

The fibroblast growth factor (FGF) signaling axis plays important roles in heart development. Yet, the molecular mechanism by which the FGF regulates cardiogenesis is not fully understood. Using genetically engineered mouse and in vitro cultured embryoid body (EB) models, we demonstrate that FGF signaling suppresses premature differentiation of heart progenitor cells, as well as autophagy in outflow tract (OFT) myocardiac cells. The FGF also promotes mesoderm differentiation in embryonic stem cells (ESCs) but inhibits cardiomyocyte differentiation of the mesoderm cells at later stages. Furthermore, inhibition of FGF signaling increases myocardial differentiation and autophagy in both ex vivo cultured embryos and EBs, whereas activation of autophagy promotes myocardial differentiation. Thus, a link between FGF signals preventing premature differentiation of heart progenitor cells and suppression of autophagy has been established. These findings provide the first evidence that autophagy plays a role in heart progenitor differentiation, and suggest a new venue to regulate stem/progenitor cell differentiation.     

The fibroblast growth factor signaling axis controls cardiac stem cell differentiation through regulating autophagy

The fibroblast growth factor (FGF) signaling axis plays important roles in heart development. Yet, the molecular mechanism by which the FGF regulates cardiogenesis is not fully understood. Using genetically engineered mouse and in vitro cultured embryoid body (EB) models, we demonstrate that FGF signaling suppresses premature differentiation of heart progenitor cells, as well as autophagy in outflow tract (OFT) myocardiac cells. The FGF also promotes mesoderm differentiation in embryonic stem cells (ESCs) but inhibits cardiomyocyte differentiation of the mesoderm cells at later stages. Furthermore, inhibition of FGF signaling increases myocardial differentiation and autophagy in both ex vivo cultured embryos and EBs, whereas activation of autophagy promotes myocardial differentiation. Thus, a link between FGF signals preventing premature differentiation of heart progenitor cells and suppression of autophagy has been established. These findings provide the first evidence that autophagy plays a role in heart progenitor differentiation, and suggest a new venue to regulate stem/progenitor cell differentiation.     

Cardiac outflow tract defects in mice lacking ALK2 in neural crest cells

Cardiac neural crest cells are multipotent migratory cells that contribute to the formation of the cardiac outflow tract and pharyngeal arch arteries. Neural crest-related developmental defects account for a large proportion of congenital heart disorders. Recently, the genetic bases for some of these disorders have been elucidated, and signaling pathways required for induction, migration and differentiation of cardiac neural crest have emerged. Bone morphogenetic proteins comprise a family of secreted ligands implicated in numerous aspects of organogenesis, including heart and neural crest development. However, it has remained generally unclear whether BMP ligands act directly on neural crest or cardiac myocytes during cardiac morphogenesis, or function indirectly by activating other cell types. Studies on BMP receptor signaling during organogenesis have been hampered by the fact that receptor knockouts often lead to early embryonic lethality. We have used a Cre/loxP system for neural crest-specific deletion of the type I receptor, ALK2, in mouse embryos. Mutant mice display cardiovascular defects, including persistent truncus arteriosus, and abnormal maturation of the aortic arch reminiscent of common forms of human congenital heart disease. Migration of mutant neural crest cells to the outflow tract is impaired, and differentiation to smooth muscle around aortic arch arteries is deficient. Moreover, in Alk2 mutants, the distal outflow tract fails to express Msx1, one of the major effectors of BMP signaling. Thus, the type I BMP receptor ALK2 plays an essential cell-autonomous role in the development of the cardiac outflow tract and aortic arch derivatives.     

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.     

Expression of cardiac neural crest and heart genes isolated by modified differential display

The invasion of the cardiac neural crest (CNC) into the outflow tract (OFT) and subsequent outflow tract septation are critical events during vertebrate heart development. We have performed four modified differential display screens in the chick embryo to identify genes that may be involved in CNC, OFT, secondary heart field, and heart development. The screens included differential display of RNA isolated from three different axial segments containing premigratory cranial neural crest cells; of RNA from distal outflow tract, proximal outflow tract, and atrioventricular tissue of embryonic chick hearts; and of RNA isolated from left and right cranial tissues, including the early heart fields. These screens have resulted in the identification of the five cDNA clones presented here, which are expressed in the cardiac neural crest, outflow tract and developing heart in patterns that are unique in heart development.     

Disheveled mediated planar cell polarity signaling is required in the second heart field lineage for outflow tract morphogenesis

Disheveled (Dvl) is a key regulator of both the canonical Wnt and the planar cell polarity (PCP) pathway. Previous genetic studies in mice indicated that outflow tract (OFT) formation requires Dvl1 and 2, but it was unclear which pathway was involved and whether Dvl1/2-mediated signaling was required in the second heart field (SHF) or the cardiac neural crest (CNC) lineage, both of which are critical for OFT development. In this study, we used Dvl1/2 null mice and a set of Dvl2 BAC transgenes that function in a pathway-specific fashion to demonstrate that Dvl1/2-mediated PCP signaling is essential for OFT formation. Lineage-specific gene-ablation further indicated that Dvl1/2 function is dispensable in the CNC, but required in the SHF for OFT lengthening to promote cardiac looping. Mutating the core PCP gene Vangl2 and non-canonical Wnt gene Wnt5a recapitulated the OFT morphogenesis defects observed in Dvl1/2 mutants. Consistent with genetic interaction studies suggesting that Wnt5a signals through the PCP pathway, Dvl1/2 and Wnt5a mutants display aberrant cell packing and defective actin polymerization and filopodia formation specifically in SHF cells in the caudal splanchnic mesoderm (SpM), where Wnt5a and Dvl2 are co-expressed specifically. Our results reveal a critical role of PCP signaling in the SHF during early OFT lengthening and cardiac looping and suggest that a Wnt5a→ Dvl PCP signaling cascade may regulate actin polymerization and protrusive cell behavior in the caudal SpM to promote SHF deployment, OFT lengthening and cardiac looping.     

Pitx2 regulates cardiac left-right asymmetry by patterning second cardiac lineage-derived myocardium

Current models of left-right asymmetry hold that an early asymmetric signal is generated at the node and transduced to lateral plate mesoderm in a linear signal transduction cascade through the function of the Nodal signaling molecule. The Pitx2 homeobox gene functions at the final stages of this cascade to direct asymmetric morphogenesis of selected organs including the heart. We previously showed that Pitx2 regulated an asymmetric pathway that was independent of cardiac looping suggesting a second asymmetric cardiac pathway. It has been proposed that in the cardiac outflow tract Pitx2 functions in both cardiac neural crest, as a target of canonical Wnt-signaling, and in the mesoderm-derived cardiac second lineage. We used fate mapping, conditional loss of function, and chimera analysis in mice to investigate the role of Pitx2 in outflow tract morphogenesis. Our findings reveal that Pitx2 is dispensable in the cardiac neural crest but functions in second lineage myocardium revealing that this cardiac progenitor field is patterned asymmetrically.     

Frs2alpha-deficiency in cardiac progenitors disrupts a subset of FGF signals required for outflow tract morphogenesis

The cardiac outflow tract (OFT) is a developmentally complex structure derived from multiple lineages and is often defective in human congenital anomalies. Although emerging evidence shows that fibroblast growth factor (FGF) is essential for OFT development, the downstream pathways mediating FGF signaling in cardiac progenitors remain poorly understood. Here, we report that FRS2alpha (FRS2), an adaptor protein that links FGF receptor kinases to multiple signaling pathways, mediates crucial aspects of FGF-dependent OFT development in mouse. Ablation of Frs2alpha in mesodermal OFT progenitor cells that originate in the second heart field (SHF) affects their expansion into the OFT myocardium, resulting in OFT misalignment and hypoplasia. Moreover, Frs2alpha mutants have defective endothelial-to-mesenchymal transition and neural crest cell recruitment into the OFT cushions, resulting in OFT septation defects. These results provide new insight into the signaling molecules downstream of FGF receptor tyrosine kinases in cardiac progenitors.     

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.     

Msx2 is an immediate downstream effector of Pax3 in the development of the murine cardiac neural crest.

The neural crest plays a crucial part in cardiac development. Cells of the cardiac subpopulation of cranial neural crest migrate from the hindbrain into the outflow tract of the heart where they contribute to the septum that divides the pulmonary and aortic channels. In Splotch mutant mice, which lack a functional Pax3 gene, migration of cardiac neural crest is deficient and aorticopulmonary septation does not occur. Downstream genes through which Pax3 regulates cardiac neural crest development are unknown. Here, using a combination of genetic and molecular approaches, we show that the deficiency of cardiac neural crest development in the Splotch mutant is caused by upregulation of Msx2, a homeobox gene with a well-documented role as a regulator of BMP signaling. We provide evidence, moreover, that Pax3 represses Msx2 expression via a direct effect on a conserved Pax3 binding site in the Msx2 promoter. These results establish Msx2 as an effector of Pax3 in cardiac neural crest development.     

BMP receptor IA is required in mammalian neural crest cells for development of the cardiac outflow tract and ventricular myocardium

The neural crest is a multipotent, migratory cell population arising from the border of the neural and surface ectoderm. In mouse, the initial migratory neural crest cells occur at the five-somite stage. Bone morphogenetic proteins (BMPs), particularly BMP2 and BMP4, have been implicated as regulators of neural crest cell induction, maintenance, migration, differentiation and survival. Mouse has three known BMP2/4 type I receptors, of which Bmpr1a is expressed in the neural tube sufficiently early to be involved in neural crest development from the outset; however, earlier roles in other domains obscure its requirement in the neural crest. We have ablated Bmpr1a specifically in the neural crest, beginning at the five-somite stage. We find that most aspects of neural crest development occur normally; suggesting that BMPRIA is unnecessary for many aspects of early neural crest biology. However, mutant embryos display a shortened cardiac outflow tract with defective septation, a process known to require neural crest cells and to be essential for perinatal viability. Surprisingly, these embryos die in mid-gestation from acute heart failure, with reduced proliferation of ventricular myocardium. The myocardial defect may involve reduced BMP signaling in a novel, minor population of neural crest derivatives in the epicardium, a known source of ventricular myocardial proliferation signals. These results demonstrate that BMP2/4 signaling in mammalian neural crest derivatives is essential for outflow tract development and may regulate a crucial proliferation signal for the ventricular myocardium.     

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. Birth Defects Research (Part C) 102:309–323, 2014. © 2014 Wiley Periodicals, Inc.     

An FGF3-BMP Signaling Axis Regulates Caudal Neural Tube Closure,Neural Crest Specification and Anterior-Posterior Axis Extension

During vertebrate axis extension, adjacent tissue layers undergo profound morphological changes: within the neuroepithelium, neural tube closure and neural crest formation are occurring, while within the paraxial mesoderm somites are segmenting from the presomitic mesoderm (PSM). Little is known about the signals between these tissues that regulate their coordinated morphogenesis. Here, we analyze the posterior axis truncation of mouse Fgf3 null homozygotes and demonstrate that the earliest role of PSM-derived FGF3 is to regulate BMP signals in the adjacent neuroepithelium. FGF3 loss causes elevated BMP signals leading to increased neuroepithelium proliferation, delay in neural tube closure and premature neural crest specification. We demonstrate that elevated BMP4 depletes PSM progenitors in vitro, phenocopying the Fgf3 mutant, suggesting that excessive BMP signals cause the Fgf3 axis defect. To test this in vivo we increased BMP signaling in Fgf3 mutants by removing one copy of Noggin, which encodes a BMP antagonist. In such mutants, all parameters of the Fgf3 phenotype were exacerbated: neural tube closure delay, premature neural crest specification, and premature axis termination. Conversely, genetically decreasing BMP signaling in Fgf3 mutants, via loss of BMP receptor activity, alleviates morphological defects. Aberrant apoptosis is observed in the Fgf3 mutant tailbud. However, we demonstrate that cell death does not cause the Fgf3 phenotype: blocking apoptosis via deletion of pro-apoptotic genes surprisingly increases all Fgf3 defects including causing spina bifida. We demonstrate that this counterintuitive consequence of blocking apoptosis is caused by the increased survival of BMP-producing cells in the neuroepithelium. Thus, we show that FGF3 in the caudal vertebrate embryo regulates BMP signaling in the neuroepithelium, which in turn regulates neural tube closure, neural crest specification and axis termination. Uncovering this FGF3-BMP signaling axis is a major advance toward understanding how these tissue layers interact during axis extension with important implications in human disease.     

Meltrin beta expressed in cardiac neural crest cells is required for ventricular septum formation of the heart

The heart is divided into four chambers by membranous septa and valves. Although evidence suggests that formation of the membranous septa requires migration of neural crest cells into the developing heart, the functional significance of these neural crest cells in the development of the endocardial cushion, an embryonic tissue that gives rise to the membranous appendages, is largely unknown. Mice defective in the protease region of Meltrin beta/ADAM19 show ventricular septal defects and defects in valve formation. In this study, by expressing Meltrin beta in either endothelial or neural crest cell lineages, we showed that Meltrin beta expressed in neural crest cells but not in endothelial cells was required for formation of the ventricular septum and valves. Although Meltrin beta-deficient neural crest cells migrated into the heart normally, they could not properly fuse the right and left ridges of the cushion tissues in the proximal outflow tract (OT), and this led to defects in the assembly of the OT and AV cushions forming the ventricular septum. These results genetically demonstrated a critical role of cardiac neural crest cells expressing Meltrin beta in triggering fusion of the proximal OT cushions and in formation of the ventricular septum.