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.     

A chemotactic model of trunk neural crest cell migration

Trunk neural crest cells follow a common ventral migratory pathway but are distributed into two distinct locations to form discrete sympathetic and dorsal root ganglia along the vertebrate axis. Although fluorescent cell labeling and time‐lapse studies have recorded complex trunk neural crest cell migratory behaviors, the signals that underlie this dynamic patterning remain unclear. The absence of molecular information has led to a number of mechanistic hypotheses for trunk neural crest cell migration. Here, we review recent data in support of three distinct mechanisms of trunk neural crest cell migration and develop and simulate a computational model based on chemotactic signaling. We show that by integrating the timing and spatial location of multiple chemotactic signals, trunk neural crest cells may be accurately positioned into two distinct targets that correspond to the sympathetic and dorsal root ganglia. In doing so, we honor the contributions of Wilhelm His to his identification of the neural crest and extend the observations of His and others to better understand a complex question in neural crest cell biology.     

Myosin-X is critical for migratory ability of Xenopus cranial neural crest cells

The neural crest is a highly migratory cell population, unique to vertebrates, that forms much of the craniofacial skeleton and peripheral nervous system. In exploring the cell biological basis underlying this behavior, we have identified an unconventional myosin, myosin-X (Myo10) that is required for neural crest migration. Myo10 is highly expressed in both premigratory and migrating cranial neural crest (CNC) cells in Xenopus embryos. Disrupting Myo10 expression using antisense morpholino oligonucleotides leads to impaired neural crest migration and subsequent cartilage formation, but only a slight delay in induction. In vivo grafting experiments reveal that Myo10-depleted CNC cells migrate a shorter distance and fail to segregate into distinct migratory streams. Finally, in vitro cultures and cell dissociation-reaggregation assays suggest that Myo10 may be critical for cell protrusion and cell-cell adhesion. These results demonstrate an essential role for Myo10 in normal cranial neural crest migration and suggest a link to cell-cell interactions and formation of processes.     

Repulsive and attractive semaphorins cooperate to direct the navigation of cardiac neural crest cells

The cardiac neural crest, a subpopulation of the neural crest, contributes to the cardiac outflow tract formation during development. However, how it follows the defined long-range migratory pathway remains unclear. We show here that the migrating cardiac neural crest cells (NCCs) express Plexin-A2, Plexin-D1 and Neuropilin. The membrane-bound ligands for Plexin-A2, Semaphorin (Sema)6A and Sema6B, are expressed in the dorsal neural tube and the lateral pharyngeal arch mesenchyme (the NCC “routes”). Sema3C, a ligand for Plexin-D1/neuropilin-1, is expressed in the cardiac outflow tract (the NCC “target”). Sema6A and Sema6B repel neural crest cells, while Sema3C attracts neural crest cells. Sema6A and Sema6B repulsion and Sema3C attraction are diminished either when Plexin-A2 and Neuropilin-1, or when Plexin-D1, respectively, are knocked down in NCCs. When RNAi knockdown diminishes each receptor in NCCs, the NCCs fail to migrate into the cardiac outflow tract in the developing chick embryo. Furthermore, Plexin-A2-deficient mice exhibit defects of cardiac outflow tract formation. We therefore conclude that the coordination of repulsive cues provided by Sema6A/Sema6B through Plexin-A2 paired with the attractive cue by Sema3C through Plexin-D1 is required for the precise navigation of migrating cardiac NCCs.     

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.     

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.     

Posteriorization by FGF, Wnt, and retinoic acid is required for neural crest induction.

The neural crest is a unique cell population induced at the lateral border of the neural plate. Neural crest is not produced at the anterior border of the neural plate, which is fated to become forebrain. Here, the roles of BMPs, FGFs, Wnts, and retinoic acid signaling in neural crest induction were analyzed by using an assay developed for investigating the posteriorization of the neural plate. Using specific markers for the anterior neural plate border and the neural crest, the posterior end of early neurula embryos was shown to be able to transform the anterior neural plate border into neural crest cells. In addition, tissue expressing anterior neural plate markers, induced by an intermediate level of BMP activity, was transformed into neural crest by posteriorizing signals. This transformation was mimicked by bFGF, Wnt-8, or retinoic acid treatment and was also inhibited by expression of the dominant negative forms of the FGF receptor, the retinoic acid receptor, and Wnt signaling molecules. The transformation of the anterior neural plate border into neural crest cells was also achieved in whole embryos, by retinoic acid treatment or by use of a constitutively active form of the retinoic acid receptor. By analyzing the expression of mesodermal markers and various graft experiments, the expression of the mutant retinoic acid receptor was shown to directly affect the ectoderm. We thereby propose a two-step model for neural crest induction. Initially, BMP levels intermediate to those required for neural plate and epidermal specification induce neural folds with an anterior character along the entire neural plate border. Subsequently, the most posterior region of this anterior neural plate border is transformed into the neural crest by the posteriorizing activity of FGFs, Wnts, and retinoic acid signals. We discuss a unifying model where lateralizing and posteriorizing signals are presented as two stages of the same inductive process required for neural crest induction.     

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.     

Vascularisation is not necessary for gut colonisation by enteric neural crest cells

The vasculature and nervous system share striking similarities in their networked, tree-like architecture and in the way they are super-imposed in mature organs. It has previously been suggested that the intestinal microvasculature network directs the migration of enteric neural crest cells (ENCC) along the gut to promote the formation of the enteric nervous system (ENS). To investigate the inter-relationship of migrating ENCC, ENS formation and gut vascular development we combined fate-mapping of ENCC with immunolabelling and intravascular dye injection to visualise nascent blood vessel networks. We found that the enteric and vascular networks initially had very distinct patterns of development. In the foregut, ENCC migrated through areas devoid of established vascular networks. In vessel-rich areas, such as the midgut and hindgut, the distribution of migrating ENCC did not support the idea that these cells followed a pre-established vascular network. Moreover, when gut vascular development was impaired, either genetically in Vegfa^120/120 or Tie2-Cre;Nrp1^fl/− mice or using an in vitro Wnt1-Cre;Rosa26^Yfp/+ mouse model of ENS development, ENCC still colonised the entire length of the gut, including the terminal hindgut. These results demonstrate that blood vessel networks are not necessary to guide migrating ENCC during ENS development. Conversely, in miRet⁵¹ mice, which lack ENS in the hindgut, the vascular network in this region appeared to be normal suggesting that in early development both networks form independently of each other.     

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.     

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.     

Cardiac neural crest contributes to cardiomyogenesis in zebrafish

In birds and mammals, cardiac neural crest is essential for heart development and contributes to conotruncal cushion formation and outflow tract septation. The zebrafish prototypical heart lacks outflow tract septation, raising the question of whether cardiac neural crest exists in zebrafish. Here, results from three distinct lineage-labeling approaches identify zebrafish cardiac neural crest cells and indicate that these cells have the ability to generate MF20-positive muscle cells in the myocardium of the major chambers during development. Fate-mapping demonstrates that cardiac neural crest cells originate both from neural tube regions analogous to those found in birds, as well as from a novel region rostral to the otic vesicle. In contrast to other vertebrates, cardiac neural crest invades the myocardium in all segments of the heart, including outflow tract, atrium, atrioventricular junction, and ventricle in zebrafish. Three distinct groups of premigratory neural crest along the rostrocaudal axis have different propensities to contribute to different segments in the heart and are correspondingly marked by unique combinations of gene expression patterns. Zebrafish will serve as a model for understanding interactions between cardiac neural crest and cardiovascular development.