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.     

Smad signaling in the neural crest regulates cardiac outflow tract remodeling through cell autonomous and non-cell autonomous effects

Neural crest cells (NCCs) are indispensable for the development of the cardiac outflow tract (OFT). Here, we show that mice lacking Smad4 in NCCs have persistent truncus arteriosus (PTA), severe OFT cushion hypoplasia, defective OFT elongation, and mispositioning of the OFT. Cardiac NCCs lacking Smad4 have increased apoptosis, apparently due to decreased Msx1/2 expression. This contributes to the reduction of NCCs in the OFT. Unexpectedly, mutants have MF20-expressing cardiomyocytes in the splanchnic mesoderm within the second heart field (SHF). This may result from abnormal differentiation or defective recruitment of differentiating SHF cells into OFT. Alterations in Bmp4, Sema3C, and PlexinA2 signals in the mutant OFT, SHF, and NCCs, disrupt the communications among different cell populations. Such disruptions can further affect the recruitment of NCCs into the OFT mesenchyme, causing severe OFT cushion hypoplasia and OFT septation failure. Furthermore, these NCCs have drastically reduced levels of Ids and MT1-MMP, affecting the positioning and remodeling of the OFT. Thus, Smad-signaling in cardiac NCCs has cell autonomous effects on their survival and non-cell autonomous effects on coordinating the movement of multiple cell lineages in the positioning and the remodeling of the OFT.     

Early regulative ability of the neuroepithelium to form cardiac neural crest

The cardiac neural crest (arising from the level of hindbrain rhombomeres 6–8) contributes to the septation of the cardiac outflow tract and the formation of aortic arches. Removal of this population after neural tube closure results in severe septation defects in the chick, reminiscent of human birth defects. Because neural crest cells from other axial levels have regenerative capacity, we asked whether the cardiac neural crest might also regenerate at early stages in a manner that declines with time. Accordingly, we find that ablation of presumptive cardiac crest at stage 7, as the neural folds elevate, results in reformation of migrating cardiac neural crest by stage 13. Fate mapping reveals that the new population derives largely from the neuroepithelium ventral and rostral to the ablation. The stage of ablation dictates the competence of residual tissue to regulate and regenerate, as this capacity is lost by stage 9, consistent with previous reports. These findings suggest that there is a temporal window during which the presumptive cardiac neural crest has the capacity to regulate and regenerate, but this regenerative ability is lost earlier than in other neural crest populations.     

Nodose placode provides ectomesenchyme to the developing chick heart in the absence of cardiac neural crest

Summary The cardiac neural crest provides both ectomesenchyme and parasympathetic postganglionic neurons to the developing heart. Ablation of cardiac neural crest results in persistent truncus arteriosus, a condition in which the conotruncal and aorticopulmonary septa do not form in the developing heart. Parasympathetic postganglionic neurons are abundantly present in hearts with persistent truncus arteriosus, which indicates a regeneration of the neural component of the cardiac neural crest without comparable restitution of the ectomesenchymal component. The neural component has been shown to be provided by cells from the nodose placode following ablation of the cardiac neural crest. This investigation has shown that ectomesenchymal cells are also supplied to a limited extent by the nodose placode which normally has no ectomesenchymal derivatives. Although placode-derived ectomesenchyme helps to strengthen the wall of the cardiac outflow vessel, it is not competent to induce conotruncal and aorticopulmonary septal closure.     

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.     

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.     

Blocking hedgehog signaling after ablation of the dorsal neural tube allows regeneration of the cardiac neural crest and rescue of outflow tract septation

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.     

Notch in the pathway: the roles of Notch signaling in neural crest development

Here, we review recent studies that suggest that Notch signaling has two roles during neural crest development: first in establishing the neural crest domain within the ectoderm via lateral induction and subsequently in diversifying the fates of cells that arise from the neural crest via lateral inhibition. The first of these roles, specification of neural crest via lateral induction, has been explored primarily in the cranial neural folds from which the cranial neural crest arises. Evidence for such a role has thus far only been obtained from chick and frog; results from these two species differ, but share the feature that Notch signaling regulates genes that are expressed by cranial neural crest through effects on expression of Bmp family members. The second of these roles, diversification of neural crest progeny via lateral inhibition, has been identified thus far only in trunk neural crest. Evidence from several species suggests that Notch-mediated lateral inhibition functions in multiple episodes in this context, in each case inhibiting neurogenesis. In the 'standard' mode of lateral inhibition, Notch promotes proliferation and in the 'instructive' mode, it promotes specific secondary fates, including cell death or glial differentiation. We raise the possibility that a single molecular mechanism, inhibition of so-called proneural bHLH genes, underlies both modes of lateral inhibition mediated by Notch signaling.     

Sonic hedgehog is required for cardiac outflow tract and neural crest cell development

The Hedgehog signaling pathway is critical for a significant number of developmental patterning events. In this study, we focus on the defects in pharyngeal arch and cardiovascular patterning present in Sonic hedgehog (Shh) null mouse embryos. Our data indicate that, in the absence of Shh, there is general failure of the pharyngeal arch development leading to cardiac and craniofacial defects. The cardiac phenotype results from arch artery and outflow tract patterning defects, as well as abnormal development of migratory neural crest cells (NCCs). The constellation of cardiovascular defects resembles a severe form of the human birth defect syndrome tetralogy of Fallot with complete pulmonary artery atresia. Previous studies have demonstrated a role for Shh in NCC survival and proliferation at later stages of development. Our data suggest that SHH signaling does not act directly on NCCs as a survival factor, but rather acts to restrict the domains that NCCs can populate during early stages (e8.5-10.5) of cardiovascular and craniofacial development.     

The right ventricle, outflow tract, and ventricular septum comprise a restricted expression domain within the secondary/anterior heart field

The vertebrate heart arises from the fusion of bilateral regions of anterior mesoderm to form a linear heart tube. Recent studies in mouse and chick have demonstrated that a second cardiac progenitor population, known as the anterior or secondary heart field, is progressively added to the heart at the time of cardiac looping. While it is clear that this second field contributes to the myocardium, its precise boundaries, other lineages derived from this population, and its contributions to the postnatal heart remain unclear. In this study, we used regulatory elements from the mouse mef2c gene to direct the expression of Cre recombinase exclusively in the anterior heart field and its derivatives in transgenic mice. By crossing these mice, termed mef2c-AHF-Cre, to Cre-dependent lacZ reporter mice, we generated a fate map of the embryonic, fetal, and postnatal heart. These studies show that the endothelial and myocardial components of the outflow tract, right ventricle, and ventricular septum are derivatives of mef2c-AHF-Cre expressing cells within the anterior heart field and its derivatives. These studies also show that the atria, epicardium, coronary vessels, and the majority of outflow tract smooth muscle are not derived from this anterior heart field population. Furthermore, a transgene marker specific for the anterior heart field is expressed in the common ventricular chamber in mef2c mutant mice, suggesting that the cardiac looping defect in these mice is not due to a failure in anterior heart field addition to the heart. Finally, the Cre transgenic mice described here will be a crucial tool for conditional gene inactivation exclusively in the anterior heart field and its derivatives.     

Homeobox b5 (Hoxb5) regulates the expression of Forkhead box D3 gene (Foxd3) in neural crest

Patterning of neural crest (NC) for the formation of specific structures along the anterio-posterior (A-P) body axis is governed by a combinatorial action of Hox genes, which are expressed in the neuroepithelium at the time of NC induction. Hoxb5 was expressed in NC at both induction and migratory stages, and our previous data suggested that Hoxb5 played a role in the NC development. However, the underlying mechanisms by which Hoxb5 regulates the early NC development are largely unknown. Current study showed that both the human and mouse Foxd3 promoters were bound and trans-activated by Hoxb5 in NC-derived neuroblastoma cells. The binding of Hoxb5 to Foxd3 promoter in vivo was further confirmed in the brain and neural tube of mouse embryos. Moreover, Wnt1-Cre mediated perturbation of Hoxb5 signaling at the dorsal neural tube in mouse embryos resulted in Foxd3 down-regulation. In ovo, Foxd3 alleviated the apoptosis of neural cells induced by perturbed Hoxb5 signaling, and Hoxb5 induced ectopic Foxd3 expression in the chick neural tube. This study demonstrated that Hoxb5 (an A-P patterning gene) regulated the NC development by directly inducing Foxd3 (a NC specifier and survival gene).     

Targeted deletion of Hand2 in cardiac neural crest-derived cells influences cardiac gene expression and outflow tract development

The basic helix-loop-helix DNA binding protein Hand2 has critical functions in cardiac development both in neural crest-derived and mesoderm-derived structures. Targeted deletion of Hand2 in the neural crest has allowed us to genetically dissect Hand2-dependent defects specifically in outflow tract and cardiac cushion independent of Hand2 functions in mesoderm-derived structures. Targeted deletion of Hand2 in the neural crest results in misalignment of the aortic arch arteries and outflow tract, contributing to development of double outlet right ventricle (DORV) and ventricular septal defects (VSD). These neural crest-derived developmental anomalies are associated with altered expression of Hand2-target genes we have identified by gene profiling. A number of Hand2 direct target genes have been identified using ChIP and ChIP-on-chip analyses. We have identified and validated a number of genes related to cell migration, proliferation/cell cycle and intracellular signaling whose expression is affected by Hand2 deletion in the neural crest and which are associated with development of VSD and DORV. Our data suggest that Hand2 is a multifunctional DNA binding protein affecting expression of target genes associated with a number of functional interactions in neural crest-derived cells required for proper patterning of the outflow tract, generation of the appropriate number of neural crest-derived cells for elongation of the conotruncus and cardiac cushion organization. Our genetic model has made it possible to investigate the molecular genetics of neural crest contributions to outflow tract morphogenesis and cell differentiation.     

Comparative gene expression analysis and fate mapping studies suggest an early segregation of cardiogenic lineages in Xenopus laevis

Retrospective clonal analysis in mice suggested that the vertebrate heart develops from two sources of cells called first and second lineages, respectively. Cells of the first lineage enter the linear heart tube and initiate terminal differentiation earlier than cells of the second lineage. It is thought that both heart lineages arise from a common progenitor cell population prior to the cardiac crescent stage (E7.5 of mouse development). The timing of segregation of different lineages as well as the molecular mechanisms underlying this process is not yet known. Furthermore, gene expression data for those lineages are very limited. Here we provide the first comparative study of cardiac marker gene expression during Xenopus laevis embryogenesis complemented by single cell RT-PCR analysis. In addition we provide fate mapping data of cardiac progenitor cells at different stages of development. Our analysis indicates an early segregation of cardiac lineages and a fairly complex heterogeneity of gene expression in the cardiac progenitor cells. Furthermore, this study sets a reference for all further studies analyzing cardiac development in X. laevis.