Molecular Pathways and Animal Models of Truncus Arteriosus.

In normal cardiovascular development in birds and mammals, the outflow tract of the heart is divided into two distinct channels to separate the oxygenated systemic blood flow from the deoxygenated pulmonary circulation. When the process of outflow tract septation fails, a single common outflow vessel persists resulting in a serious clinical condition known as persistent truncus arteriosus or common arterial trunk. In this chapter, we will review molecular pathways and the cells that are known to play a role in the formation and development of the outflow tract and how genetic manipulation of these pathways in animal models can result in common arterial trunk.

Human Genetics of Truncus Arteriosus.

Integrated human genetics and molecular/developmental biology studies have revealed that truncus arteriosus is highly associated with 22q11.2 deletion syndrome. Other congenital malformation syndromes and variants in genes encoding TBX, GATA, and NKX transcription factors and some signaling proteins have also been reported as its etiology.

NKX2-6 related congenital heart disease: Biallelic homeodomain-disrupting variants and truncus arteriosus.

Congenital heart defects (CHD) are the most common birth defect and are both clinically and genetically heterogeneous. Truncus arteriosus (TA), characterized by a single arterial vessel arising from both ventricles giving rise to the coronary, pulmonary and systemic arteries, is rare and only responsible for 1% of all CHD. Two consanguineous families with TA were previously identified to have homozygous nonsense variants within the gene NKX2-6. NKX2-6 is a known downstream target of TBX1, an important transcriptional regulator implicated in the cardiac phenotype of 22q11.2 microdeletion syndrome. Herein, we report two siblings with TA presumably caused by compound heterozygous NKX2-6 variants without a history of consanguinity. Two in-house cohorts with conotruncal defects (CTD) were sequenced for variants in NKX2-6 and no additional cases of biallelic NKX2-6 variants were identified. The similar phenotype of these cases, and the clustering of variants that likely result in a truncated protein that disrupts the homeobox domain, suggest that biallelic loss of function for NKX2-6 is a rare genetic etiology for TA in particular, and possibly other types of CHD.

Gata4 regulates hedgehog signaling and Gata6 expression for outflow tract development.

Dominant mutations of Gata4, an essential cardiogenic transcription factor (TF), were known to cause outflow tract (OFT) defects in both human and mouse, but the underlying molecular mechanism was not clear. In this study, Gata4 haploinsufficiency in mice was found to result in OFT defects including double outlet right ventricle (DORV) and ventricular septum defects (VSDs). Gata4 was shown to be required for Hedgehog (Hh)-receiving progenitors within the second heart field (SHF) for normal OFT alignment. Restored cell proliferation in the SHF by knocking-down Pten failed to rescue OFT defects, suggesting that additional cell events under Gata4 regulation is important. SHF Hh-receiving cells failed to migrate properly into the proximal OFT cushion, which is associated with abnormal EMT and cell proliferation in Gata4 haploinsufficiency. The genetic interaction of Hh signaling and Gata4 is further demonstrated to be important for OFT development. Gata4 and Smo double heterozygotes displayed more severe OFT abnormalities including persistent truncus arteriosus (PTA). Restoration of Hedgehog signaling renormalized SHF cell proliferation and migration, and rescued OFT defects in Gata4 haploinsufficiency. In addition, there was enhanced Gata6 expression in the SHF of the Gata4 heterozygotes. The Gata4-responsive repressive sites were identified within 1kbp upstream of the transcription start site of Gata6 by both ChIP-qPCR and luciferase reporter assay. These results suggested a SHF regulatory network comprising of Gata4, Gata6 and Hh-signaling for OFT development.

Reduced dosage of ß-catenin provides significant rescue of cardiac outflow tract anomalies in a Tbx1 conditional null mouse model of 22q11.2 deletion syndrome.

The 22q11.2 deletion syndrome (22q11.2DS; velo-cardio-facial syndrome; DiGeorge syndrome) is a congenital anomaly disorder in which haploinsufficiency of TBX1, encoding a T-box transcription factor, is the major candidate for cardiac outflow tract (OFT) malformations. Inactivation of Tbx1 in the anterior heart field (AHF) mesoderm in the mouse results in premature expression of pro-differentiation genes and a persistent truncus arteriosus (PTA) in which septation does not form between the aorta and pulmonary trunk. Canonical Wnt/ß-catenin has major roles in cardiac OFT development that may act upstream of Tbx1. Consistent with an antagonistic relationship, we found the opposite gene expression changes occurred in the AHF in ß-catenin loss of function embryos compared to Tbx1 loss of function embryos, providing an opportunity to test for genetic rescue. When both alleles of Tbx1 and one allele of ß-catenin were inactivated in the Mef2c-AHF-Cre domain, 61% of them (n = 34) showed partial or complete rescue of the PTA defect. Upregulated genes that were oppositely changed in expression in individual mutant embryos were normalized in significantly rescued embryos. Further, ß-catenin was increased in expression when Tbx1 was inactivated, suggesting that there may be a negative feedback loop between canonical Wnt and Tbx1 in the AHF to allow the formation of the OFT. We suggest that alteration of this balance may contribute to variable expressivity in 22q11.2DS.

46,XY disorders of sex development and congenital diaphragmatic hernia: a case with dysmorphic facies, truncus arteriosus, bifid thymus, gut malrotation, rhizomelia, and adactyly.

The association of 46,XY disorder of sex development (DSD) with congenital diaphragmatic hernia (CDH) is rare, but has been previously described with and without other congenital anomalies. Literature review identified five cases of 46,XY DSD associated with CDH and other congenital anomalies. These five cases share characteristics including CDH, 46,XY karyotype with external female appearing or ambiguous genitalia, cardiac anomalies, and decreased life span. The present case had novel features including truncus arteriosus, bifid thymus, gut malrotation, and limb anomalies consisting of rhizomelia and adactyly. With this case report, we present a review of the literature of cases of 46,XY DSD and CDH in association with multiple congenital abnormalities. This case may represent a unique syndrome of 46,XY DSD and diaphragmatic hernia or a more severe presentation of a syndrome represented in the previously reported cases.

Ontogeny of vessel wall components in the outflow tract of the chick.

During development of the outflow tract, the walls of the truncus arteriosus change from a diffuse extracellular matrix (ECM) surrounded by an extension of the myocardium to alternating laminae of smooth muscle and elastic connective tissue. The transition rapidly follows septation, when mesenchyme associated with the endothelium differentiates. Using immunocytochemical methods with antibodies to components of the tunica media and the tunica adventitia we have analysed the differentiation of the vessel walls of the outflow tract of the chick. The tunica media marker, elastin, forms laminae in a radial sequence, beginning at the outer margin of the truncus mesenchyme. Conversely, smooth muscle myosin is first expressed in cells associated with the endothelium. Laminin is expressed as a cell surface component throughout the development of the outflow tract. Matrix fibronectin distribution is correlated with the regions that will form the tunica media and apparently forms a radial gradient which is highest near the endothelium. Markers for the tunica adventitia, collagen I and VI, are expressed first at the peripheries of the newly formed tunica media, and collagen VI expression spreads radially through the tunica media. Thus, the vessel wall components appear within the mesenchyme of the truncus arteriosus in opposed radial gradients of differentiation. The tunica media cells acquire secretory and contractile phenotypes independently and may be responding to different stimuli in their expression of these features.

Decrease in calcitonin-containing cells in truncus arteriosus.

Experimental studies in chick embryos have demonstrated that truncus arteriosus (TA), a form of conotruncal cardiac defect, is due to abnormalities in the cranial neural crest. However, no data are available to support this hypothesis in humans with isolated TA. In the present study, the assessment of calcitonin immunoreactive cells (C-cells) has been employed to evaluate whether or not the proportion of thyroid cells derived from the cranial neural crest is normal in patients with isolated TA. Thyroid sections from 15 such patients in which no other extracardiac malformations were neither clinically nor pathologically found, and from 11 control age-matched patients were studied immunohistochemically at autopsy in order to determine the number and distribution of calcitonin-containing cells. The volume density of C-cells (0.888%) and the number of C-cells per follicle (0.991) was significantly lower in patients with TA than in control patients (3.475%, and 2.367, respectively). The decrease of neural crest-derived cells in the thyroid of patients with "isolated" TA documents more extent abnormalities than clinically suspected and supports the hypothesis of neural crest disturbance as the pathogenetic factor responsible for this heart malformation.

Development of the musculoelastic septation complex in the avian truncus arteriosus.

It is now well established that cells from the cardiac neural crest (CNC) are essential for normal conotruncal septation. The truncal septation complex consists of the aorticopulmonary (AP) septum and the myocardial sheath of the truncus. The principal role of the CNC cells during septation appears to be their differentiation into the elastogenic smooth muscle that forms the AP septum proper. The objective of this study was to integrate serial reconstruction and specific histochemical markers in order to provide a unified analysis of the relationships between the CNC and the other components of the truncal septation complex. The development of the septation complex was compared normal embryos vs. embryos from which the CNC had been surgically ablated. Embryos from each group were harvested after incubation periods of 4-8 days (Hamburger-Hamilton stages 23-34). Histochemical procedures were performed for positive identification of the elastic matrix and smooth muscle alpha-actin; the presence of these proteins was used as the criterion for "septal cells" and to define the boundaries of the septum. The results indicate that the shape, components, boundaries, and degree of organization of the septation complex may be different from previous descriptions. Furthermore, all of the components of the truncal septation complex are dysgenic in the absence of the CNC. Of special significance in the absence of CNC. Of special significance in the absence of CNC are: 1) the failure of the myocardial sheath to retract; 2) the apparently random distribution of surrogate ectomesenchyme; and 3) the impairment of truncal elastogenesis. These results indicate that the cells of neural crest origin interact with the surrounding mesenchyme during septation and that the entire septation complex depends upon the presence of the neural crest cells for normal development.

Distribution of vitronectin in the embryonic chick heart during endocardial cell migration.

In the early phase of heart development, the endocardial cells migrate into the truncal swellings and atrioventricular (AV) cushions, and become mesenchymal cells. Vitronectin is a glycoprotein which is thought to mediate cell migration. The present study demonstrates by immunohistochemistry the distribution of vitronectin in order to elucidate its contribution to endocardial cell migration in the developing chick heart. At Hamburger and Mamilton's stage 23, the network of fibrillar material filled the extracellular space of both truncal swellings and AV cushions. The fibrillar network has been thought to be a matrix for endocardial cell migration. The network was stained with the anti-vitronectin antibody. At stage 29, the swellings and cushions were packed with mesenchymal cells, though immunoreactivity to the antibody was still observed in the extracellular matrix. The myocardium facing the AV cushions reacted to the antibody, but the myocardium surrounding the truncus arteriosus did not. The intensity of the immunohistochemical staining of the myocardium facing the AV cushions increased and reached a peak at stages 24 to 26, and then became weak by stage 29. The endocardial sheet, aortico-pulmonary septum and developing tunica media of the great arteries were not stained by the antibody at any stage. These results strongly suggest that vitronectin is involved in the migration of endocardial cells, and that the myocardium facing the AV cushions produces vitronectin.

Peripheral development of the avian vagus nerve with special reference to the morphological innervation of heart and lung.

The development of the cardio-pulmonary innervation was studied in whole-mount specimens of chick embryos stained with the anti-neurofilament protein (NFP) antibody. From the morphological point of view, vagal branches could be classified into two categories, i.e., the branchial branches primarily related to the pharyngeal arches, and intestinal arborization derivatives which are associated primarily with the primitive gut. The former consisted of the superior cardiac branch innervating the truncus arteriosus of the heart, and the latter, the sinal branch, pulmonary branches as well as recurrent nerve and the other intestinal branches. The superior cardiac branch at first develops as a pair of branchial branches which passes into the truncus arteriosus at stage 25, and later rotates along the aortic arch 6, thus making an asymmetrical configuration by stage 27. The sinal branch is a medial branch which first develops at stage 24. It arises from the junction of each intestinal arborization in close association with the pulmonary branch.

Origin and propagation of elastogenesis in the developing cardiovascular system.

Ectomesenchyme derived from cardiac neural crest is critical to aorticopulmonary septation in the heart. However, any unique contribution of the cardiac ectomesenchyme to the extracellular matrix of the conotruncus has not been demonstrated previously. In this study the chronology and topography of soluble tropoelastin (STE) and the aldehyde-rich protein (ARP) of the elastic connective tissues have been examined in the chick embryo, stages 21-38, and in the quail-chick chimera, stages 24-35 (quail neural fold grafted onto a chick embryo). STE was located with immunofluorescence histochemistry, and ARP with Schiff's reagent. With these procedures prevenient sites of elastin synthesis are observed readily. The results show that the myocardium proper appears to have a role in the instigation of elastogenesis and in elastic fiber orientation; that the mesenchymal cells whose matrix contains elastic fibers are ectomesenchymal, of neural crest origin; and that elastin is deployed in an orderly proximal-distal sequence. It is hypothesized that elastogenesis is a critical event in aorticopulmonary septation.

Distribution of fibronectin during the morphogenesis of the truncus.

The distribution of fibronectin during morphogenesis of the truncus has been studied in chick embryo hearts between days 4 and 10 of incubation. Fibronectin appears as a natural marker for some truncal cells and permits the visualization of the different processes of cell rearrangement which result in truncal morphogenesis. The mesenchymal cells involved in formation of the aorto-pulmonary septum and in formation of the arterial tunica media are intensely fluorescent for fibronectin. These cells present fibrillar deposits of fibronectin associated with their surfaces. Very little staining for fibronectin is observed in association with the anlage of the semilunar valves. Prospective adventitial cells are negative for fibronectin. The close association between the fluorescence and the surface of the cells involved in formation of the aorto-pulmonary septum and the arterial tunica media suggests that fibronectin may be implicated in the formation of both structures. The intensity and amount of the fluorescence staining decreases as morphogenesis of the truncus is completed. The decrease in fibronectin staining is suggested to be related to changes in cell phenotypic expression.

Morphogenesis of the truncus arteriosus of the chick embryo heart: tissue reorganization during septation.

Septation of the truncus arteriosus of the normal chick embryo heart was surveyed systematically with the light microscope. Tissue from replicate samples at successive periods of development was sectioned within an arbitrary coordinate system based on positional reference points along the external surface of the heart. Correlation of several aspects of tissue morphology within this spatial and temporal reference-frame yielded a new description of tissue associations and kinetics during septation. A stable complex of tissue structures appeared in the downstream, distal truncus at Stage 25 and persisted throughout the septation process. This complex consisted of (1) the cephalic margin of the myocardial sheath, and (2) the adjacent bifurcation of the vascular lumen, linked together by (3) the newly condensed Y-shaped strap of cells forming the aorticopulmonary septum. The apparent motion of this septation-complex toward the ventricle(s), the appearance within the thoracic cavity of the adjacent segments of the aortic arches, and measures of tissue length and width suggested that septation was accompanied, and perhaps initiated, by increased tension along the truncus. The truncal ridges remained upstream from the complex, with mesenchymal condensations beneath the endocardium differentiating into the definitive semilunar valves. Downstream from the bifurcation, mesenchyme in the aortic arch region condensed around the separate lumens to form the smooth muscular tunica media of the great arteries. The epicardium developed in a caudocephalic direction along the heart tube. Vagal innervation approached the heart cephalocaudally. Capillaries formed along the dividing truncus in both directions. Autoradiography following 3H-thymidine labelling demonstrated reduced DNA synthetic activity in the cephalic margin of the myocardium and aorticopulmonary septum, compared with the associated loose mesenchyme.