Over-expression of Fgf8 in cardiac neural crest cells leads to persistent truncus arteriosus.

During cardiogenesis, the outflow tract undergoes a complicated morphogenesis, including the re-alignment of the great blood vessels, and the separation of aorta and pulmonary trunk. The deficiency of FGF8 in the morphogenesis of outflow tract has been well studied, however, the effect of over-dosed FGF8 on the development of outflow tract remains unknown. In this study, Rosa26R-Fgf8 knock-in allele was constitutively activated by Wnt1-cre transgene in the mouse neural crest cells presumptive for the endocardial cushion of outflow tract. Surprisingly, Wnt1-cre; Rosa26R-Fgf8 mouse embryos exhibited persistent truncus arteriosus and died prior to E15.5. The cardiac neural crest cells in Wnt1-cre; Rosa26R-Fgf8 truncus arteriosus did not degenerate as in WT controls, but proliferated into a thickened endocardial cushion and then, blocked the blood outflow from cardiac chambers into the lungs, which resulted in the embryonic lethality. Although the spiral aorticopulmonary septum failed to form, the differentiaion of the endothelium and smooth muscle in the Wnt1-cre; Rosa26R-Fgf8 truncus arteriosus were impacted little. However, lineage tracing assay showed that the neural crest derived cells aggregated in the cushion layer, but failed to differentiate into the endothelium of Wnt1-cre; Rosa26R-Fgf8 truncus arteriosus. Further investigation displayed the reduced p-Akt and p-Erk immunostaining, and the decreased Bmp2 and Bmp4 transcription in the endothelium of Wnt1-cre; Rosa26R-Fgf8 truncus arteriosus. Our findings suggested that Fgf8 over-expression in cardiac neural crest impaired the formation of aorticopulmonary septum by suppressing the endothelial differentiation and stimulating the proliferation of endocardial cushion cells, which implicated a novel etiology of persistent truncus arteriosus.

Inositol 1,4,5-trisphosphate receptor 2 as a novel marker of vasculature to delineate processes of cardiopulmonary development.

Congenital heart diseases (CHDs) involving the outflow tract (OFT), such as persistent truncus arteriosus (PTA), lead to mortality and morbidity with implications not only in the heart, but also in the pulmonary vasculature. The mechanisms of pulmonary artery (PA) development and the etiologies underlying PA disorders associated with CHD remain poorly understood partly because of a specific marker for PA development is nonexistent. The three subtypes of inositol 1,4,5-trisphosphate receptors (IP3R1, 2, and 3) are intracellular Ca2+ channels that are essential for many tissues and organs. We discovered that IP3R2 was expressed in the vasculature and heart during development using transgenic mice, in which a LacZ marker gene was knocked into the IP3R2 locus. Whole-mount and section LacZ staining showed that IP3R2-LacZ-positive cells were detectable exclusively in the smooth muscle cells, or tunica media, of PA, merging into αSMA-positive cells during development. Furthermore, our analyses suggested that IP3R2-LacZ positive PA smooth muscle layers gradually elongate from the central PA to the peripheral PAs from E13.5 to E18.5, supporting the distal angiogenesis theory for the development of PA, whereas IP3R2-LacZ was rarely expressed in smooth muscle cells in the pulmonary trunk. Crossing IP3R-LacZ mice with mice hypomorphic for Tbx1 alleles revealed that PTA of Tbx1 mutants may result from agenesis or hypoplasia of the pulmonary trunk; thus, the left and right central to peripheral PAs connect directly to the dorsal side of the truncus arteriosus in these mutants. Additionally, we found hypercellular interstitial mesenchyme and delayed maturation of the lung endoderm in the Tbx1 mutant lungs. Our study identifies IP3R2 as a novel marker for clear visualization of PA during development and can be utilized for studying cardiopulmonary development and disease.

ASK1 mediates the teratogenicity of diabetes in the developing heart by inducing ER stress and inhibiting critical factors essential for cardiac development.

Maternal diabetes in mice induces heart defects similar to those observed in human diabetic pregnancies. Diabetes enhances apoptosis and suppresses cell proliferation in the developing heart, yet the underlying mechanism remains elusive. Apoptosis signal-regulating kinase 1 (ASK1) activates the proapoptotic c-Jun NH2-terminal kinase 1/2 (JNK1/2) leading to apoptosis, suggesting a possible role of ASK1 in diabetes-induced heart defects. We aimed to investigate whether ASK1 is activated in the heart and whether deleting the Ask1 gene blocks diabetes-induced adverse events and heart defect formation. The ASK1-JNK1/2 pathway was activated by diabetes. Deleting Ask1 gene significantly reduced the rate of heart defects, including ventricular septal defects (VSDs) and persistent truncus arteriosus (PTA). Additionally, Ask1 deletion diminished diabetes-induced JNK1/2 phosphorylation and its downstream transcription factors and endoplasmic reticulum (ER) stress markers. Consistent with this, caspase activation and apoptosis were blunted. Ask1 deletion blocked the increase in cell cycle inhibitors (p21 and p27) and the decrease in cyclin D1 and D3 and reversed diabetes-repressed cell proliferation. Ask1 deletion also restored the expression of BMP4, NKX2.5, and GATA5, Smad1/5/8 phosphorylation, whose mutations or deletion result in reduced cell proliferation, VSD, and PTA formation. We conclude that ASK1 may mediate the teratogenicity of diabetes through activating the JNK1/2-ER stress pathway and inhibiting cell cycle progression, thereby impeding the cardiogenesis pathways essential for ventricular septation and outflow tract development.

Congenital diseases and semaphorin signaling: overview to date of the evidence linking them.

Semaphorins and their receptors, neuropilins and plexins, were initially characterized as a modulator of axonal guidance during development, but are now recognized as a regulator of a wide range of developmental events including morphogenesis and angiogenesis, and activities of the immune system. Owing to the development of next-generation sequencing technologies together with other useful DNA assays, it has also become clear that semaphorin signaling plays a crucial role in many congenital diseases such as retinal degeneration and congenital heart defects. This review summarizes the recent knowledge about the relationship between a variety of congenital diseases and semaphorin signaling.

Bmp signaling represses Vegfa to promote outflow tract cushion development.

Congenital heart disease (CHD) is a devastating anomaly that affects ∼1% of live births. Defects of the outflow tract (OFT) make up a large percentage of human CHD. We investigated Bmp signaling in mouse OFT development by conditionally deleting both Bmp4 and Bmp7 in the second heart field (SHF). SHF Bmp4/7 deficiency resulted in defective epithelial to mesenchymal transition (EMT) and reduced cardiac neural crest ingress, with resultant persistent truncus arteriosus. Using a candidate gene approach, we found that Vegfa was upregulated in the Bmp4/7 mutant hearts. To determine if Vegfa is a downstream Bmp effector during EMT, we examined whether Vegfa is transcriptionally regulated by the Bmp receptor-regulated Smad. Our findings indicate that Smad directly binds to Vegfa chromatin and represses Vegfa transcriptional activity. We also found that Vegfa is a direct target for the miR-17-92 cluster, which is also regulated by Bmp signaling in the SHF. Deletion of miR-17-92 reveals similar phenotypes to Bmp4/7 SHF deletion. To directly address the function of Vegfa repression in Bmp-mediated EMT, we performed ex vivo explant cultures from Bmp4/7 and miR-17-92 mutant hearts. EMT was defective in explants from the Bmp4/7 double conditional knockout (dCKO; Mef2c-Cre;Bmp4/7(f/f)) and miR-17-92 null. By antagonizing Vegfa activity in explants, EMT was rescued in Bmp4/7 dCKO and miR-17-92 null culture. Moreover, overexpression of miR-17-92 partially suppressed the EMT defect in Bmp4/7 mutant embryos. Our study reveals that Vegfa levels in the OFT are tightly controlled by Smad- and microRNA-dependent pathways to modulate OFT development.

Outflow tract cushions perform a critical valve-like function in the early embryonic heart requiring BMPRIA-mediated signaling in cardiac neural crest.

Neural crest-specific ablation of BMP type IA receptor (BMPRIA) causes embryonic lethality by embryonic day (E) 12.5, and this was previously postulated to arise from a myocardial defect related to signaling by a small population of cardiac neural crest cells (cNCC) in the epicardium. However, as BMP signaling via cNCC is also required for proper development of the outflow tract cushions, precursors to the semilunar valves, a plausible alternate or additional hypothesis is that heart failure may result from an outflow tract cushion defect. To investigate whether the outflow tract cushions may serve as dynamic valves in regulating hemodynamic function in the early embryo, in this study we used noninvasive ultrasound biomicroscopy-Doppler imaging to quantitatively assess hemodynamic function in mouse embryos with P0-Cre transgene mediated neural crest ablation of Bmpr1a (P0 mutants). Similar to previous studies, the neural crest-deleted Bmpr1a P0 mutants died at approximately E12.5, exhibiting persistent truncus arteriosus, thinned myocardium, and congestive heart failure. Surprisingly, our ultrasound analyses showed normal contractile indices, heart rate, and atrioventricular conduction in the P0 mutants. However, reversed diastolic arterial blood flow was detected as early as E11.5, with cardiovascular insufficiency and death rapidly ensuing by E12.5. Quantitative computed tomography showed thinning of the outflow cushions, and this was associated with a marked reduction in cell proliferation. These results suggest BMP signaling to cNCC is required for growth of the outflow tract cushions. This study provides definitive evidence that the outflow cushions perform a valve-like function critical for survival of the early mouse embryo.

Differences in extra-cellular matrix and myocyte homeostasis between the neonatal right ventricle in hypoplastic left heart syndrome and truncus arteriosus.

OBJECTIVE: The right ventricle in hypoplastic left heart syndrome (HLHS) works at systemic pressure and large volume loading before and after first stage palliation. There is a paucity of information regarding the intrinsic characteristics of the right ventricle in HLHS. We studied extra-cellular matrix composition, myocyte homeostasis and gene expression in right ventricular biopsies obtained from patients with HLHS undergoing neonatal first stage palliation and from patients undergoing neonatal truncus arteriosus repair. METHODS: Tissue was evaluated using histological and real-time PCR techniques using the truncus group as a comparative group. Mean difference in outcomes between the HLHS and truncus groups was estimated using linear regression models in unadjusted and age-adjusted analyses. RESULTS: Markers of cell proliferation, apoptosis and fibronectin were significantly higher in the right ventricular myocardium of patients with hypoplastic left heart syndrome compared to truncus arteriosus. Type I collagen content and NKX2.5 expression were significantly lower in HLHS than the truncus group. CONCLUSION: The neonatal right ventricle in HLHS demonstrates a number of intrinsic differences compared to the right ventricle in truncus arteriosus including relative immaturity of the extra-cellular matrix, inappropriately low transcription factor expression and increased myocyte apoptosis.

Maternal diabetes induces congenital heart defects in mice by altering the expression of genes involved in cardiovascular development.

BACKGROUND: Congenital heart defects are frequently observed in infants of diabetic mothers, but the molecular basis of the defects remains obscure. Thus, the present study was performed to gain some insights into the molecular pathogenesis of maternal diabetes-induced congenital heart defects in mice. METHODS AND RESULTS: We analyzed the morphological changes, the expression pattern of some genes, the proliferation index and apoptosis in developing heart of embryos at E13.5 from streptozotocin-induced diabetic mice. Morphological analysis has shown the persistent truncus arteriosus combined with a ventricular septal defect in embryos of diabetic mice. Several other defects including defective endocardial cushion (EC) and aberrant myofibrillogenesis have also been found. Cardiac neural crest defects in experimental embryos were analyzed and validated by the protein expression of NCAM and PGP 9.5. In addition, the protein expression of Bmp4, Msx1 and Pax3 involved in the development of cardiac neural crest was found to be reduced in the defective hearts. The mRNA expression of Bmp4, Msx1 and Pax3 was significantly down-regulated (p < 0.001) in the hearts of experimental embryos. Further, the proliferation index was significantly decreased (p < 0.05), whereas the apoptotic cells were significantly increased (p < 0.001) in the EC and the ventricular myocardium of the experimental embryos. CONCLUSION: It is suggested that the down-regulation of genes involved in development of cardiac neural crest could contribute to the pathogenesis of maternal diabetes-induced congenital heart defects.

Normal fate and altered function of the cardiac neural crest cell lineage in retinoic acid receptor mutant embryos.

Mouse embryos lacking the retinoic acid (RA) receptors RARalpha1 and RARbeta suffer from a failure to properly septate (divide) the early outflow tract of the heart into distinct aortic and pulmonary channels, a phenotype termed persistent truncus arteriosus. This phenotype is associated with a failure in the development of the cardiac neural crest cell lineage, which normally forms the aorticopulmonary septum. In this study, we examined the fate of the neural crest lineage in RARalpha1/RARbeta mutant embryos by crossing with the Wnt1-cre and conditional R26R alleles, which together constitute a genetic lineage marker for the neural crest. We find that the number, migration, and terminal fate of the cardiac neural crest is normal in mutant embryos; however, the specific function of these cells in forming the aorticopulmonary septum is impaired. We furthermore show that the neural crest cells themselves do not utilize retinoid receptors and do not respond to RA during this process, but rather that the phenotype is cell non-autonomous for the neural crest cell lineage. This suggests that an alternative tissue in the vicinity of the outflow tract of the heart responds directly to RA, and thereby induces or permits the neural crest cell lineage to initiate aorticopulmonary septation.

Calcium currents in hearts with persistent truncus arteriosus.

We have used the neural crest model of defective heart development to characterize both L- and T-type Ca2+ currents (ICa,L and ICa,T) in ventricular myocytes from embryonic chick hearts with a severe outflow tract anomaly known as persistent truncus arteriosus (PTA). Because of smaller whole embryo weights but no significant change in the weights of ventricles with PTA, the ventricle to whole embryo weight ratios from hearts with PTA were 61% larger than normal at day 11 of incubation. There was a 51% reduction in the peak magnitude of ICa,L at a test potential of +10 mV (-1.4 vs. -0.7 microA/cm2), whereas ICa,T, a proportionately large fraction of the total Ca2+ current in the embryonic chick ventricle, was unaffected. In comparison to sham-operated controls, ICa,L was otherwise not different. Half-activation occurred at about -1 and -41 mV, whereas half-inactivation occurred at -19 and -61 mV for ICa,L and ICa,T, respectively. The time for half-recovery from inactivation were not different and were 200 and 230 ms for ICa,L and ICa,T, respectively. The time for half-decay of the currents and their responses to BAY K 8644 were also similar in both sham-operated and experimental hearts. Although the dihydropyridine receptor binding experiments suggest that the total number of L-type Ca2+ channels was not different, the results from the physiological experiments indicate that the number of functional L-type channels available for opening and/or the single-channel conductance may be reduced in hearts with PTA. Finally, our results with the neural crest model indicate that it is unlikely that the development of Ca2+ currents is influenced by the onset of cholinergic innervation in the heart.

Spatial disorder of collagens in the great vessels, associated with congenital heart defects.

Surgical ablation of the cardiac neural crest from the chicken embryo results in persistent truncus arteriosus (PTA) and a change in the elastic laminae of the great vessels, wherein elastin and the elastin microfibril show significant spatial disorder. The purpose of this study was to test the hypothesis that the interstitial collagens would also be disordered in the elastic laminae of chicken embryos with PTA. The birefringence characteristics of interstitial collagen were examined to evaluate spatial ordering. The results showed that collagen in the elastic laminae assumed an orderly configuration of well-defined fiber bundles in the great vessel walls of control embryos, whereas vessels from embryos with PTA lacked any distinct spatial order. Collagens type I and III were localized in the vessel walls. Type III collagen was the principal collagen of the elastic laminae, but was absent from the intima of all vessels. In the elastic laminae of vessels from control embryos, collagen type III showed well-defined fiber bundles whereas embryos with PTA had diffuse collagen type III in poorly defined laminae that were not separated by discrete layers of smooth muscle cells. Collagen type I was a minor component of the elastic laminae but formed robust pericellular fiber bundles throughout the media and intima. Collagen type I fibers appeared to be coarsened and less uniform in the vessels from embryos with PTA.