Single-cell transcriptomics uncovers a non-autonomous Tbx1-dependent genetic program controlling cardiac neural crest cell development.

Disruption of cardiac neural crest cells (CNCCs) results in congenital heart disease, yet we do not understand the cell fate dynamics as these cells differentiate to vascular smooth muscle cells. Here we performed single-cell RNA-sequencing of NCCs from the pharyngeal apparatus with the heart in control mouse embryos and when Tbx1, the gene for 22q11.2 deletion syndrome, is inactivated. We uncover three dynamic transitions of pharyngeal NCCs expressing Tbx2 and Tbx3 through differentiated CNCCs expressing cardiac transcription factors with smooth muscle genes. These transitions are altered non-autonomously by loss of Tbx1. Further, inactivation of Tbx2 and Tbx3 in early CNCCs results in aortic arch branching defects due to failed smooth muscle differentiation. Loss of Tbx1 interrupts mesoderm to CNCC cell-cell communication with upregulation and premature activation of BMP signaling and reduced MAPK signaling, as well as alteration of other signaling, and failed dynamic transitions of CNCCs leading to disruption of aortic arch artery formation and cardiac outflow tract septation.

Embryonic aortic arch material properties obtained by optical coherence tomography-guided micropipette aspiration.

It is challenging to determine the in vivo material properties of a very soft, mesoscale arterial vesselsof size âˆ¼ 80 to 120 µm diameter. This information is essential to understand the early embryonic cardiovascular development featuring rapidly evolving dynamic microstructure. Previous research efforts to describe the properties of the embryonic great vessels are very limited. Our objective is to measure the local material properties of pharyngeal aortic arch tissue of the chick-embryo during the early Hamburger-Hamilton (HH) stages, HH18 and HH24. Integrating the micropipette aspiration technique with optical coherence tomography (OCT) imaging, a clear vision of the aspirated arch geometry is achieved for an inner pipette radius of Rp = 25 µm. The aspiration of this region is performed through a calibrated negatively pressurized micro-pipette. A computational finite element model is developed to model the nonlinear behaviour of the arch structure by considering the geometry-dependent constraints. Numerical estimations of the nonlinear material parameters for aortic arch samples are presented. The exponential material nonlinearity parameter (a) of aortic arch tissue increases statistically significantly from a = 0.068 ± 0.013 at HH18 to a = 0.260 ± 0.014 at HH24 (p = 0.0286). As such, the aspirated tissue length decreases from 53 µm at HH18 to 34 µm at HH24. The calculated NeoHookean shear modulus increases from 51 Pa at HH18 to 93 Pa at HH24 which indicates a statistically significant stiffness increase. These changes are due to the dynamic changes of collagen and elastin content in the media layer of the vessel during development.

Whole-Mount Immunofluorescence Protocol for 3D Imaging, Reconstruction, and Quantification of Fourth Pharyngeal Arch Artery Formation.

Pharyngeal arch arteries (PAA) are formed early during mouse embryogenesis and remodel soon thereafter into the aortic arch arteries. Failure of these vessels to form or remodel results in congenital heart defects. This protocol is designed to study the formation of the PAA using whole-mount immunofluorescence staining, followed by tissue clearing with benzyl alcohol/benzyl benzoate (BAAB) and imaging by confocal microscopy. The fine cellular resolution obtained with this technique allows the embryonic vasculature of the pharyngeal arch artery endothelium to be visualized by surface rendering and quantitatively analyzed by counting the number of endothelial cells in both the PAA and the vascular plexus surrounding them.

Pharyngeal pouches provide a niche microenvironment for arch artery progenitor specification.

The paired pharyngeal arch arteries (PAAs) are transient blood vessels connecting the heart with the dorsal aorta during embryogenesis. Although PAA malformations often occur along with pharyngeal pouch defects, the functional interaction between these adjacent tissues remains largely unclear. Here, we report that pharyngeal pouches are essential for PAA progenitor specification in zebrafish embryos. We reveal that the segmentation of pharyngeal pouches coincides spatiotemporally with the emergence of PAA progenitor clusters. These pouches physically associate with pharyngeal mesoderm in discrete regions and provide a niche microenvironment for PAA progenitor commitment by expressing BMP proteins. Specifically, pouch-derived BMP2a and BMP5 are the primary niche cues responsible for activating the BMP/Smad pathway in pharyngeal mesoderm, thereby promoting progenitor specification. In addition, BMP2a and BMP5 play an inductive function in the expression of the cloche gene npas4l in PAA progenitors. cloche mutants exhibit a striking failure to specify PAA progenitors and display ectopic expression of head muscle markers in the pharyngeal mesoderm. Therefore, our results support a crucial role for pharyngeal pouches in establishing a progenitor niche for PAA morphogenesis via BMP2a/5 expression.

Visualization and Analysis of Pharyngeal Arch Arteries using Whole-mount Immunohistochemistry and 3D Reconstruction.

Improper formation or remodeling of the pharyngeal arch arteries (PAAs) 3, 4, and 6 contribute to some of the most severe forms of congenital heart disease. To study the formation of PAAs, we developed a protocol using whole-mount immunofluorescence coupled with benzyl alcohol/benzyl benzoate (BABB) tissue clearing, and confocal microscopy. This allows for the visualization of the pharyngeal arch endothelium at a fine cellular resolution as well as the 3D connectivity of the vasculature. Using software, we have established a protocol to quantify the number of endothelial cells (ECs) in PAAs, as well as the number of ECs within the vascular plexus surrounding the PAAs within pharyngeal arches 3, 4, and 6. When applied to the whole embryo, this methodology provides a comprehensive visualization and quantitative analysis of embryonic vasculature.

Importance of endothelial Hey1 expression for thoracic great vessel development and its distal enhancer for Notch-dependent endothelial transcription.

Thoracic great vessels such as the aorta and subclavian arteries are formed through dynamic remodeling of embryonic pharyngeal arch arteries (PAAs). Previous work has shown that loss of a basic helix-loop-helix transcription factor Hey1 in mice causes abnormal fourth PAA development and lethal great vessel anomalies resembling congenital malformations in humans. However, how Hey1 mediates vascular formation remains unclear. In this study, we revealed that Hey1 in vascular endothelial cells, but not in smooth muscle cells, played essential roles for PAA development and great vessel morphogenesis in mouse embryos. Tek-Cre-mediated Hey1 deletion in endothelial cells affected endothelial tube formation and smooth muscle differentiation in embryonic fourth PAAs and resulted in interruption of the aortic arch and other great vessel malformations. Cell specificity and signal responsiveness of Hey1 expression were controlled through multiple cis-regulatory regions. We found two distal genomic regions that had enhancer activity in endothelial cells and in the pharyngeal epithelium and somites, respectively. The novel endothelial enhancer was conserved across species and was specific to large-caliber arteries. Its transcriptional activity was regulated by Notch signaling in vitro and in vivo, but not by ALK1 signaling and other transcription factors implicated in endothelial cell specificity. The distal endothelial enhancer was not essential for basal Hey1 expression in mouse embryos but may likely serve for Notch-dependent transcriptional control in endothelial cells together with the proximal regulatory region. These findings help in understanding the significance and regulation of endothelial Hey1 as a mediator of multiple signaling pathways in embryonic vascular formation.

Pax9 is required for cardiovascular development and interacts with Tbx1 in the pharyngeal endoderm to control 4th pharyngeal arch artery morphogenesis.

Developmental defects affecting the heart and aortic arch arteries are a significant phenotype observed in individuals with 22q11 deletion syndrome and are caused by a microdeletion on chromosome 22q11. TBX1, one of the deleted genes, is expressed throughout the pharyngeal arches and is considered a key gene, when mutated, for the arch artery defects. Pax9 is expressed in the pharyngeal endoderm and is downregulated in Tbx1 mutant mice. We show here that Pax9-deficient mice are born with complex cardiovascular malformations that affect the outflow tract and aortic arch arteries with failure of the 3rd and 4th pharyngeal arch arteries to form correctly. Transcriptome analysis indicated that Pax9 and Tbx1 may function together, and mice double heterozygous for Tbx1/Pax9 presented with a significantly increased incidence of interrupted aortic arch when compared with Tbx1 heterozygous mice. Using a novel Pax9Cre allele, we demonstrated that the site of this Tbx1-Pax9 genetic interaction is the pharyngeal endoderm, therefore revealing that a Tbx1-Pax9-controlled signalling mechanism emanating from the pharyngeal endoderm is required for crucial tissue interactions during normal morphogenesis of the pharyngeal arch artery system.

Isl-1 positive pharyngeal mesenchyme subpopulation and its role in the separation and remodeling of the aortic sac in embryonic mouse heart.

BACKGROUND: Second heart field cells and neural crest cells have been reported to participate in the morphogenesis of the pharyngeal arch arteries (PAAs); however, how the PAAs grow out and are separated from the aortic sac into left and right sections is unknown. RESULTS: An Isl-1 positive pharyngeal mesenchyme protrusion in the aortic sac ventrally extends and fuses with the aortic sac wall to form a midsagittal septum that divides the aortic sac. The aortic sac division separates the left and right PAAs to form independent arteries. The midsagittal septum dividing the aortic sac has a different expression pattern from the aortic-pulmonary (AP) septum in which Isl-1 positive cells are absent. At 11 days post-conception (dpc) in a mouse embryo, the Isl-1 positive mesenchyme protrusion appears as a heart-shaped structure, in which subpopulations with Isl-1+ Tbx3+ and Isl-1+ Nkx2.5+ cells are included. CONCLUSIONS: The aortic sac is a dynamic structure that is continuously divided during the migration from the pharyngeal mesenchyme to the pericardial cavity. The separation of the aortic sac is not complete until the AP septum divides the aortic sac into the ascending aorta and pulmonary trunk. Moreover, the midsagittal septum and the AP septum are distinct structures.

The ubiquitin ligase HECTD1 promotes retinoic acid signaling required for development of the aortic arch.

The development of the aortic arch is a complex process that involves remodeling of the bilaterally symmetrical pharyngeal arch arteries (PAAs) into the mature asymmetric aortic arch. Retinoic acid signaling is a key regulator of this process by directing patterning of the second heart field (SHF), formation of the caudal PAAs and subsequent remodeling of the PAAs to form the aortic arch. Here, we identify the HECTD1 ubiquitin ligase as a novel modulator of retinoic acid signaling during this process. Hectd1opm/opm homozygous mutant embryos show a spectrum of aortic arch abnormalities that occur following loss of 4th PAAs and increased SHF marker expression. This sequence of defects is similar to phenotypes observed in mutant mouse models with reduced retinoic acid signaling. Importantly, HECTD1 binds to and influences ubiquitination of the retinoic acid receptor, alpha (RARA). Furthermore, reduced activation of a retinoic acid response element (RARE) reporter is detected in Hectd1 mutant cells and embryos. Interestingly, Hectd1opm/+ heterozygous embryos exhibit reduced retinoic acid signaling, along with intermediate increased expression of SHF markers; however, heterozygotes show normal development of the aortic arch. Decreasing retinoic acid synthesis by reducing Raldh2 (also known as Aldh1a2) gene dosage in Hectd1opm/+ heterozygous embryos reveals a genetic interaction. Double heterozygous embryos show hypoplasia of the 4th PAA and increased incidence of a benign aortic arch variant, in which the transverse arch between the brachiocephalic and left common carotid arteries is shortened. Together, our data establish that HECTD1 is a novel regulator of retinoic acid signaling required for proper aortic arch development.

Surgical Strategy for and Anatomic Locations of Petroapex and Petroclival Meningiomas Based on Evaluation of the Feeding Artery.

BACKGROUND: The anterior transpetrosal (ATP) approach is the most appropriate approach for petroclival meningiomas (PCMs), which are typically located from the dorsum sellae to the upper border of the internal auditory meatus (IAM). Although neurosurgeons can resect over this area if the tumor is detached from the dura, tumors within the indication area for PCMs are not appropriate for the ATP approach, because it can be difficult to evaluate whether the tumor is attached to or only touching the dura. In this study, we investigated the tumor extension area based on an evaluation of the feeding artery to achieve a more accurate assessment of the dural attachment area. METHODS: Using various angiography techniques, we studied 51 feeding arteries from 24 patients who had undergone surgical treatment of primary petroapex meningiomas and PCMs via the ATP approach. We measured the lower and posterior extension distances, the extension rate of the cavernous sinus and Meckel's cave, and the midline extension rate of the tumors. RESULTS: The ascending pharyngeal artery (AphA) was the predominant feeding artery for tumors with lower extension. We determined that tumors extending over the lower border of IAM in cases in which the feeding artery was not the AphA can be resected using the ATP approach. CONCLUSIONS: This study shows an association between the predominant feeding artery and tumor extension area and demonstrates that an evaluation of the dural attachment area based on the feeding artery can aid selection of the appropriate surgical approach.

TGF-ß Signaling Is Necessary and Sufficient for Pharyngeal Arch Artery Angioblast Formation.

The pharyngeal arch arteries (PAAs) are transient embryonic blood vessels that mature into critical segments of the aortic arch and its branches. Although defects in PAA development cause life-threating congenital cardiovascular defects, the molecular mechanisms that orchestrate PAA morphogenesis remain unclear. Through small-molecule screening in zebrafish, we identified TGF-ß signaling as indispensable for PAA development. Specifically, chemical inhibition of the TGF-ß type I receptor ALK5 impairs PAA development because nkx2.5+ PAA progenitor cells fail to differentiate into tie1+ angioblasts. Consistent with this observation, we documented a burst of ALK5-mediated Smad3 phosphorylation within PAA progenitors that foreshadows angioblast emergence. Remarkably, premature induction of TGF-ß receptor activity stimulates precocious angioblast differentiation, thereby demonstrating the sufficiency of this pathway for initiating the PAA progenitor to angioblast transition. More broadly, these data uncover TGF-ß as a rare signaling pathway that is necessary and sufficient for angioblast lineage commitment.

The pro-inflammatory signalling regulator Stat4 promotes vasculogenesis of great vessels derived from endothelial precursors.

Vasculogenic defects of great vessels (GVs) are a major cause of congenital cardiovascular diseases. However, genetic regulators of endothelial precursors in GV vasculogenesis remain largely unknown. Here we show that Stat4, a transcription factor known for its regulatory role of pro-inflammatory signalling, promotes GV vasculogenesis in zebrafish. We find stat4 transcripts highly enriched in nkx2.5+ endothelial precursors in the pharynx and demonstrate that genetic ablation of stat4 causes stenosis of pharyngeal arch arteries (PAAs) by suppressing PAAs 3-6 angioblast development. We further show that stat4 is a downstream target of nkx2.5 and that it autonomously promotes proliferation of endothelial precursors of the mesoderm. Mechanistically, stat4 regulates the emerging PAA angioblasts by inhibiting the expression of hdac3 and counteracting the effect of stat1a. Altogether, our study establishes a role for Stat4 in zebrafish great vessel development, and suggests that Stat4 may serve as a therapeutic target for GV defects.

Blood flow patterns underlie developmental heart defects.

Although cardiac malformations at birth are typically associated with genetic anomalies, blood flow dynamics also play a crucial role in heart formation. However, the relationship between blood flow patterns in the early embryo and later cardiovascular malformation has not been determined. We used the chicken embryo model to quantify the extent to which anomalous blood flow patterns predict cardiac defects that resemble those in humans and found that restricting either the inflow to the heart or the outflow led to reproducible abnormalities with a dose-response type relationship between blood flow stimuli and the expression of cardiac phenotypes. Constricting the outflow tract by 10-35% led predominantly to ventricular septal defects, whereas constricting by 35-60% most often led to double outlet right ventricle. Ligation of the vitelline vein caused mostly pharyngeal arch artery malformations. We show that both cardiac inflow reduction and graded outflow constriction strongly influence the development of specific and persistent abnormal cardiac structure and function. Moreover, the hemodynamic-associated cardiac defects recapitulate those caused by genetic disorders. Thus our data demonstrate the importance of investigating embryonic blood flow conditions to understand the root causes of congenital heart disease as a prerequisite to future prevention and treatment.NEW & NOTEWORTHY Congenital heart defects result from genetic anomalies, teratogen exposure, and altered blood flow during embryonic development. We show here a novel "dose-response" type relationship between the level of blood flow alteration and manifestation of specific cardiac phenotypes. We speculate that abnormal blood flow may frequently underlie congenital heart defects.

Pharyngeal arch artery defects and lethal malformations of the aortic arch and its branches in mice deficient for the Hrt1/Hey1 transcription factor.

The aortic arch and major branch arteries are formed from the three pairs of pharyngeal arch arteries (PAAs) during embryonic development. Their morphological defects are clinically observed as isolated diseases, as a part of complicated cardiovascular anomalies or as a manifestation of multi-organ syndromes such as 22q11.2 deletion syndrome. Although numerous genes have been implicated in PAA formation and remodeling, detailed mechanisms remain poorly understood. Here we report that the mice null for Hrt1/Hey1, a gene encoding a downstream transcription factor of Notch and ALK1 signaling pathways, show perinatal lethality on the C57BL/6N, C57BL/6N × C57BL/6J or C57BL/6N × 129X1/SvJ background. Hrt1/Hey1 null embryos display abnormal development of the fourth PAA (PAA4), which results in congenital vascular defects including right-sided aortic arch, interruption of the aortic arch and aberrant origin of the right subclavian artery. Impaired vessel formation occurs randomly in PAA4 of Hrt1/Hey1 null embryos, which likely causes the variability of congenital malformations. Endothelial cells in PAA4 of null embryos differentiate normally but are structurally disorganized at embryonic day 10.5 and 11.5. Vascular smooth muscle cells are nearly absent in the structurally-defective PAA4, despite the appropriate migration of cardiac neural crest cells into the fourth pharyngeal arches. Endothelial expression of Jag1 is down-regulated in the structurally-defective PAA4 of null embryos, which may be one of the mechanisms underlying the suppression of vascular smooth muscle cell differentiation. While the direct downstream phenomena of the Hrt1/Hey1 deficiency remain to be clarified, we suggest that Hrt1/Hey1-dependent transcriptional regulation has an important role in PAA formation during embryonic development.

Postotic and preotic cranial neural crest cells differently contribute to thyroid development.

Thyroid development and formation vary among species, but in most species the thyroid morphogenesis consists of five stages: specification, budding, descent, bilobation and folliculogenesis. The detailed mechanisms of these stages have not been fully clarified. During early development, the cranial neural crest (CNC) contributes to the thyroid gland. The removal of the postotic CNC (corresponding to rhombomeres 6, 7 and 8, also known as the cardiac neural crest) results in abnormalities of the cardiovascular system, thymus, parathyroid glands, and thyroid gland. To investigate the influence of the CNC on thyroid bilobation process, we divided the CNC into two regions, the postotic CNC and the preotic CNC (from the mesencephalon to rhombomere 5) regions and examined. We found that preotic CNC-ablated embryos had a unilateral thyroid lobe, and confirmed the presence of a single lobe or the absence of lobes in postotic CNC-ablated chick embryos. The thyroid anlage in each region-ablated embryos was of a normal size at the descent stage, but at a later stage, the thyroid in preotic CNC-ablated embryos was of a normal size, conflicting with a previous report in which the thyroid was reduced in size in the postotic CNC-ablated embryos. The postotic CNC cells differentiated into connective tissues of the thyroid in quail-to-chick chimeras. In contrast, the preotic CNC cells did not differentiate into connective tissues of the thyroid. We found that preotic CNC cells encompassed the thyroid anlage from the specification stage to the descent stage. Finally, we found that endothelin-1 and endothelin type A receptor-knockout mice and bosentan (endothelin receptor antagonist)-treated chick embryos showed bilobation anomalies that included single-lobe formation. Therefore, not only the postotic CNC, but also the preotic CNC plays an important role in thyroid morphogenesis.

Neural crest cells in cardiovascular development.

Cardiac neural crest cells (NCCs) are a transient, migratory cell population exclusive to vertebrate embryos. Ablation, transplantation, and lineage-tracing experiments in chick and mouse have demonstrated their essential role in the remodeling of the initially bilateral and symmetric pharyngeal artery pairs into an aortic arch and for the septation of the cardiac outflow tract into the base of the pulmonary artery and aorta. Accordingly, defective cardiac NCC function is a common cause of congenital birth defects. Here, we review our current understanding of cardiac NCC-mediated vascular remodeling and signaling pathways important for this process. We additionally discuss their contribution to the cardiac valves as well as the still contentious role of cardiac NCCs in the development of the myocardium and conductive system of the heart.

The peptide hormone cholecystokinin modulates the tonus and compliance of the bulbus arteriosus and pre-branchial vessels of the rainbow trout (Oncorhynchus mykiss).

The bulbus arteriosus is a compliant structure between the ventricle and ventral aorta of teleost fish. It serves as a "wind-kessel" that dampens pressure variations during the cardiac cycle allowing a continuous flow of blood into the gills. The bulbus arteriosus receives sympathetic innervation and is affected by several circulating substances, indicating neurohumoral control. We have previously shown that the peptide hormone, cholecystokinin (CCK), affects the hemodynamics of the cardiovascular system in rainbow trout (Oncorhynchus mykiss) by increasing flow pulse amplitude without affecting cardiac output. We hypothesized that this could be explained by an altered tonus or compliance/distensibility of the bulbus arteriosus. Our results show that there is a substantial effect of CCK on the bulbus arteriosus. Concentrations of CCK that altered the cardiac function of in situ perfused hearts also contracted the bulbus arteriosus in vitro. Pressure-volume curves revealed a change in both the tonus and the compliance/distensibility of this structure. Furthermore, the stimulatory (constricting) effect of CCK was also evident in the ventricle and vasculature leading to the gills, but absent in the atrium, efferent branchial arteries and dorsal aorta. In conclusion, CCK alters the mechanical properties of the ventricle, bulbus arteriosus, ventral aorta and afferent gill vasculature, thus maintaining adequate branchial and systemic blood flow and pressure when cardiorespiratory demands change, such as after feeding.

Prdm1 functions in the mesoderm of the second heart field, where it interacts genetically with Tbx1, during outflow tract morphogenesis in the mouse embryo.

Congenital heart defects affect at least 0.8% of newborn children and are a major cause of lethality prior to birth. Malformations of the arterial pole are particularly frequent. The myocardium at the base of the pulmonary trunk and aorta and the arterial tree associated with these great arteries are derived from splanchnic mesoderm of the second heart field (SHF), an important source of cardiac progenitor cells. These cells are controlled by a gene regulatory network that includes Fgf8, Fgf10 and Tbx1. Prdm1 encodes a transcriptional repressor that we show is also expressed in the SHF. In mouse embryos, mutation of Prdm1 affects branchial arch development and leads to persistent truncus arteriosus (PTA), indicative of neural crest dysfunction. Using conditional mutants, we show that this is not due to a direct function of Prdm1 in neural crest cells. Mutation of Prdm1 in the SHF does not result in PTA, but leads to arterial pole defects, characterized by mis-alignment or reduction of the aorta and pulmonary trunk, and abnormalities in the arterial tree, defects that are preceded by a reduction in outflow tract size and loss of caudal pharyngeal arch arteries. These defects are associated with a reduction in proliferation of progenitor cells in the SHF. We have investigated genetic interactions with Fgf8 and Tbx1, and show that on a Tbx1 heterozygote background, conditional Prdm1 mutants have more pronounced arterial pole defects, now including PTA. Our results identify PRDM1 as a potential modifier of phenotypic severity in TBX1 haploinsufficient DiGeorge syndrome patients.

Development of the human aortic arch system captured in an interactive three-dimensional reference model.

Variations and mutations in the human genome, such as 22q11.2 microdeletion, can increase the risk for congenital defects, including aortic arch malformations. Animal models are increasingly expanding our molecular and genetic insights into aortic arch development. However, in order to justify animal-to-human extrapolations, a human morphological, and molecular reference model would be of great value, but is currently lacking. Here, we present interactive three-dimensional reconstructions of the developing human aortic arch system, supplemented with the protein distribution of developmental markers for patterning and growth, including T-box transcription factor TBX1, a major candidate for the phenotypes found in patients with the 22q11.2 microdeletion. These reconstructions and expression data facilitate unbiased interpretations, and reveal previously unappreciated aspects of human aortic arch development. Based on our reconstructions and on reported congenital anomalies of the pulmonary trunk and tributaries, we postulate that the pulmonary arteries originate from the aortic sac, rather than from the sixth pharyngeal arch arteries. Similar to mouse, TBX1 is expressed in pharyngeal mesenchyme and epithelia. The endothelium of the pharyngeal arch arteries is largely negative for TBX1 and family member TBX2 but expresses neural crest marker AP2α, which gradually decreases with ongoing development of vascular smooth muscle. At early stages, the pharyngeal arch arteries, aortic sac, and the dorsal aortae in particular were largely negative for proliferation marker Ki67, potentially an important parameter during aortic arch system remodeling. Together, our data support current animal-to-human extrapolations and future genetic and molecular analyses using animal models of congenital heart disease. © 2013 Wiley Periodicals, Inc.