Targeted disruption of semaphorin 3C leads to persistent truncus arteriosus and aortic arch interruption.

Semaphorin 3C is a secreted member of the semaphorin gene family. To investigate its function in vivo, we have disrupted the semaphorin 3C locus in mice by targeted mutagenesis. semaphorin 3C mutant mice die within hours after birth from congenital cardiovascular defects consisting of interruption of the aortic arch and improper septation of the cardiac outflow tract. This phenotype is similar to that reported following ablation of the cardiac neural crest in chick embryos and resembles congenital heart defects seen in humans. Semaphorin 3C is expressed in the cardiac outflow tract as neural crest cells migrate into it. Their entry is disrupted in semaphorin 3C mutant mice. These data suggest that semaphorin 3C promotes crest cell migration into the proximal cardiac outflow tract.     

Fibronectin and integrin alpha 5 play essential roles in the development of the cardiac neural crest

Cardiac neural crest (CNC) plays a requisite role during cardiovascular development and defects in the formation of CNC-derived structures underlie several common forms of human congenital birth defects. Migration of the CNC cells to their destinations as well as expansion and maintenance of these cells are important for the normal development of the cardiac outflow tract and aortic arch arteries; however, molecular mechanisms regulating these processes are not well-understood. Fibronectin (FN) protein is present along neural crest migration paths and neural crest cells migrate when plated on FN in vitro; therefore, we tested the role of FN during the development of the CNC in vivo. Our analysis of the fate of the neural crest shows that CNC cells reach their destinations in the branchial arches and the cardiac outflow tract in the absence of FN or its cellular receptor integrin α5β1. However, we found that FN and integrin α5 modulate CNC proliferation and survival, and are required for the presence of normal numbers of CNC cells at their destinations.     

The outflow tract of the heart in fishes: anatomy, genes and evolution

A large number of congenital heart defects associated with mortality in humans are those that affect the cardiac outflow tract, and this provides a strong imperative to understand its development during embryogenesis. While there is wide phylogenetic variation in adult vertebrate heart morphology, recent work has demonstrated evolutionary conservation in the early processes of cardiogenesis, including that of the outflow tract. This, along with the utility and high reproductive potential of fish species such as Danio rerio , Oryzias latipes etc. , suggests that fishes may provide ideal comparative biological models to facilitate a better understanding of this poorly understood region of the heart. In this review, the authors present the current understanding of both phylogeny and ontogeny of the cardiac outflow tract in fishes and examine how new molecular studies are informing the phylogenetic relationships and evolutionary trajectories that have been proposed. The authors also attempt to address some of the issues of nomenclature that confuse this area of research.     

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 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.     

BMP signaling is required for septation of the outflow tract of the mammalian heart

Bone morphogenetic proteins (BMPs) constitute a family of approximately 20 growth factors involved in a tremendous variety of embryonic inductive processes. BMPs elicit dose-dependent effects on patterning during gastrulation and gradients of BMP activity are thought to be established through regulation of the relative concentrations of BMP receptors, ligands and antagonists. We tested whether later developmental events also are sensitive to reduced levels of BMP signaling. We engineered a knockout mouse that expresses a BMP type II receptor that lacks half of the ligand-binding domain. This altered receptor is expressed at levels comparable with the wild-type allele, but has reduced signaling capability. Unlike Bmpr2-null mice, mice homozygous for this hypomorphic receptor undergo normal gastrulation, providing genetic evidence of the dose-dependent effects of BMPs during mammalian development. Mutants, however, die at midgestation with cardiovascular and skeletal defects, demonstrating that the development of these tissues requires wild-type levels of BMP signaling. The most striking defects occur in the outflow tract of the heart, with absence of septation of the conotruncus below the valve level and interrupted aortic arch, a phenotype known in humans as persistent truncus arteriosus (type A4). In addition, semilunar valves do not form in mutants, while the atrioventricular valves appear unaffected. Abnormal septation of the heart and valve anomalies are the most frequent forms of congenital cardiac defects in humans; however, most mouse models display broad defects throughout cardiac tissues. The more restricted spectrum of cardiac anomalies in Bmpr2(deltaE2) mutants makes this strain a key murine model to understand the embryonic defects of persistent truncus arteriosus and impaired semilunar valve formation in humans.     

Computational hemodynamic optimization predicts dominant aortic arch selection is driven by embryonic outflow tract orientation in the chick embryo

In the early embryo, a series of symmetric, paired vessels, the aortic arches, surround the foregut and distribute cardiac output to the growing embryo and fetus. During embryonic development, the arch vessels undergo large-scale asymmetric morphogenesis to form species-specific adult great vessel patterns. These transformations occur within a dynamic biomechanical environment, which can play an important role in the development of normal arch configurations or the aberrant arch morphologies associated with congenital cardiac defects. Arrested migration and rotation of the embryonic outflow tract during late stages of cardiac looping has been shown to produce both outflow tract and several arch abnormalities. Here, we investigate how changes in flow distribution due to a perturbation in the angular orientation of the embryonic outflow tract impact the morphogenesis and growth of the aortic arches. Using a combination of in vivo arch morphometry with fluorescent dye injection and hemodynamics-driven bioengineering optimization-based vascular growth modeling, we demonstrate that outflow tract orientation significantly changes during development and that the associated changes in hemodynamic load can dramatically influence downstream aortic arch patterning. Optimization reveals that balancing energy expenditure with diffusive capacity leads to multiple arch vessel patterns as seen in the embryo, while minimizing energy alone led to the single arch configuration seen in the mature arch of aorta. Our model further shows the critical importance of the orientation of the outflow tract in dictating morphogenesis to the adult single arch and accurately predicts arch IV as the dominant mature arch of aorta. These results support the hypothesis that abnormal positioning of the outflow tract during early cardiac morphogenesis may lead to congenital defects of the great vessels due to altered hemodynamic loading.     

Notch signaling in cardiac valve development and disease

The Notch pathway is an intercellular signaling mechanism involved in multiple cell-to-cell communication processes that regulate cell fate specification, differentiation, and tissue patterning during embryogenesis and adulthood. Functional studies in the mouse have shown that a Hey-Bmp2 regulatory circuit restricts Bmp2 expression to presumptive valve myocardium (atrioventricular canal and outflow tract). Likewise, a Notch-Hey-Bmp2 axis represses Bmp2 in the endocardium. During cardiac valve formation, endocardial Notch signaling activates the epithelial-mesenchyme transition (EMT) that will give rise to the cardiac valve primordia. During this process, Notch integrates with myocardially derived signals (Bmp2 or Bmp4) to promote, via Snail1/2 activation a complete, invasive EMT in presumptive valve tissue. In humans, mutations in Notch signaling components are associated with several congenital disorders involving malformed valves, aortic arch, and defective chamber septation. Data suggest that the same embryonic Notch-Hey-Bmp2 regulatory axis is active in the adult valve. This review examines the experimental evidence supporting a role for Notch in heart valve development and homeostasis, and how altered Notch signaling may lead to valve disease in the newborn and adult.     

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.     

Connexin 43 expression reflects neural crest patterns during cardiovascular development

We used transgenic mice in which the promoter sequence for connexin 43 linked to a lacZ reporter was expressed in neural crest but not myocardial cells to document the pattern of cardiac neural crest cells in the caudal pharyngeal arches and cardiac outflow tract. Expression of lacZ was strikingly similar to that of cardiac neural crest cells in quail-chick chimeras. By using this transgenic mouse line to compare cardiac neural crest involvement in cardiac outflow septation and aortic arch artery development in mouse and chick, we were able to note differences and similarities in their cardiovascular development. Similar to neural crest cells in the chick, lacZ-positive cells formed a sheath around the persisting aortic arch arteries, comprised the aorticopulmonary septation complex, were located at the site of final fusion of the conal cushions, and populated the cardiac ganglia. In quail-chick chimeras generated for this study, neural crest cells entered the outflow tract by two pathways, submyocardially and subendocardially. In the mouse only the subendocardial population of lacZ-positive cells could be seen as the cells entered the outflow tract. In addition lacZ-positive cells completely surrounded the aortic sac prior to septation, while in the chick, neural crest cells were scattered around the aortic sac with the bulk of cells distributed in the bridging portion of the aorticopulmonary septation complex. In the chick, submyocardial populations of neural crest cells assembled on opposite sides of the aortic sac and entered the conotruncal ridges. Even though the aortic sac in the mouse was initially surrounded by lacZ-positive cells, the two outflow vessels that resulted from its septation showed differential lacZ expression. The ascending aorta was invested by lacZ-positive cells while the pulmonary trunk was devoid of lacZ staining. In the chick, both of these vessels were invested by neural crest cells, but the cells arrived secondarily by displacement from the aortic arch arteries during vessel elongation. This may indicate a difference in derivation of the pulmonary trunk in the mouse or a difference in distribution of cardiac neural crest cells. An independent mouse neural crest marker is needed to confirm whether the differences are indeed due to species differences in cardiovascular and/or neural crest development. Nevertheless, with the differences noted, we believe that this mouse model faithfully represents the location of cardiac neural crest cells. The similarities in location of lacZ-expressing cells in the mouse to that of cardiac neural crest cells in the chick suggest that this mouse is a good model for studying mammalian cardiac neural crest and that the mammalian cardiac neural crest performs functions similar to those shown for chick.     

Fgf15 is required for proper morphogenesis of the mouse cardiac outflow tract

Evidence in animal models indicates that signaling networks functioning in the developing pharyngeal arches regulate stereotyped processes critical for proper development of the aortic arch and cardiac outflow tract. Here, we describe the phenotype of mice lacking fibroblast growth factor 15 (Fgf15), which encodes a secreted signaling molecule expressed within the developing pharyngeal arches. Homozygous Fgf15 mutants present heart defects consistent with malalignment of the aorta and pulmonary trunk. These defects correlate with early morphological defects of the outflow tract due to aberrant behavior of the cardiac neural crest. We demonstrate that Fgf15 expression within the pharyngeal arches is unaltered by a loss of Tbx1, a key regulator of pharyngeal arch development implicated in DiGeorge syndrome. In addition, Fgf15 and Tbx1 do not interact genetically, suggesting that Fgf15 operates through a pathway independent of Tbx1. These studies reveal a novel role of Fgf15 during development of the cardiac outflow tract.     

DiGeorge syndrome and pharyngeal apparatus development

DiGeorge syndrome is the most frequent microdeletion syndrome in humans, and is characterized by cardiovascular, thymic and parathyroid, and craniofacial anomalies. The underlying cause is disturbed formation of the pharyngeal apparatus, a transient structure present during vertebrate development that gives rise to endocrine glands, craniofacial tissue, and the cardiac outflow tract. The pharyngeal apparatus is composed of derivatives of ectoderm, endoderm, mesoderm and the neural crest. Thus, complex interactions between cell types from different origins have to be orchestrated in the correct spatiotemporal manner to establish proper formation of the pharyngeal system. The analysis of engineered mouse mutants developing a phenotype resembling DiGeorge syndrome has revealed genes and signalling pathways crucial for this process. Intriguingly, these mouse models reveal that interference with either of two distinct phases of pharyngeal apparatus development can contribute to the aetiology of DiGeorge syndrome.     

Elevated transforming growth factor beta2 enhances apoptosis and contributes to abnormal outflow tract and aortic sac development in retinoic X receptor alpha knockout embryos

Septation of the single tubular embryonic outflow tract into two outlet segments in the heart requires the precise integration of proliferation, differentiation and apoptosis during remodeling. Lack of proper coordination between these processes would result in a variety of congenital cardiac defects such as those seen in the retinoid X receptor alpha knockout (Rxra(-/-)) mouse. Rxra(-/-) embryos exhibit lethality between embryonic day (E) 13.5 and 15.5 and harbor a variety of conotruncal and aortic sac defects making it an excellent system to investigate the molecular and morphogenic causes of these cardiac malformations. At E12.5, before the embryonic lethality, we found no qualitative difference between wild type and Rxra(-/-) proliferation (BrdU incorporation) in outflow tract cushion tissue but a significant increase in apoptosis as assessed by both TUNEL labeling in paraffin sections and caspase activity in trypsin-dispersed hearts. Additionally, E12.5 embryos demonstrated elevated levels of transforming growth factor beta2 (TGFbeta2) protein in multiple cell lineages in the heart. Using a whole-mouse-embryo culture system, wild-type E11.5 embryos treated with TGFbeta2 protein for 24 hours displayed enhanced apoptosis in both the sinistroventralconal cushion and dextrodorsalconal cushion in a manner analogous to that observed in the Rxra(-/-). TGFbeta2 protein treatment also led to malformations in both the outflow tract and aortic sac. Importantly, Rxra(-/-) embryos that were heterozygous for a null mutation in the Tgfb2 allele exhibited a partial restoration of the elevated apoptosis and of the malformations. This was evident at both E12.5 and E13.5. The data suggests that elevated levels of TGFbeta2 can (1) contribute to abnormal outflow tract morphogenesis by enhancing apoptosis in the endocardial cushions and (2) promote aortic sac malformations by interfering with the normal development of the aorticopulmonary septum.     

Fgf8 expression in the Tbx1 domain causes skeletal abnormalities and modifies the aortic arch but not the outflow tract phenotype of Tbx1 mutants

Fgf8 and Tbx1 have been shown to interact in patterning the aortic arch, and both genes are required in formation and growth of the outflow tract of the heart. However, the nature of the interaction of the two genes is unclear. We have utilized a novel Tbx1(Fgf8) allele which drives Fgf8 expression in Tbx1-positive cells and an inducible Cre-LoxP recombination system to address the role of Fgf8 in Tbx1 positive cells in modulating cardiovascular development. Results support a requirement of Fgf8 in Tbx1 expressing cells to finely control patterning of the aortic arch and great arteries specifically during the pharyngeal arch artery remodeling process and indicate that the endoderm is the most likely site of this interaction. Furthermore, our data suggest that Fgf8 and Tbx1 play independent roles in regulating outflow tract development. This finding is clinically relevant since TBX1 is the candidate for DGS/VCFS, characterized clinically by variable expressivity and reduced penetrance of cardiovascular defects; Fgf8 gene variants may provide molecular clues to this variability.