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Introduction: Neural crest cells (NCCs) are multipotent and are attributed to the combination of complex multimodal gene regulatory mechanisms. Cardiac neural crest (CNC) cells, originating from the dorsal neural tube, are pivotal architects of the cardio-neuro-vascular domain, which orchestrates the embryogenesis of critical cardiac and vascular structures. Remarkably, while the scientific community compiled a comprehensive inventory of neural crest derivatives by the early 1980s, our understanding of the CNC's role in various cardiovascular disease processes still needs to be explored. This review delves into the differentiation of NCC, specifically the CNC cells, and explores the diverse facets of non-syndromic cardiovascular neurocristopathies., Methods: A systematic review was conducted as per the PRISMA Statement. Three prominent databases, PubMed, Scopus, and Embase, were searched, which yielded 1,840 studies. We excluded 1,796 studies, and the final selection of 44 studies formed the basis of this comprehensive review., Results: Neurocristopathies are a group of genetic disorders that affect the development of cells derived from the NC. Cardiovascular neurocristopathy, i.e., cardiopathy and vasculopathy, associated with the NCC could occur in the form of (1) cardiac septation disorders, mainly the aortico-pulmonary septum; (2) great vessels and vascular disorders; (3) myocardial dysfunction; and (4) a combination of all three phenotypes. This could result from abnormalities in NCC migration, differentiation, or proliferation leading to structural abnormalities and are attributed to genetic, familial, sporadic or acquired causes., Discussion: Phenotypic characteristics of cardiovascular neurocristopathies, such as bicuspid aortic valve and thoracic aortic aneurysm, share a common embryonic origin and are surprisingly prevalent in the general population, necessitating further research to identify the underlying pathogenic and genetic factors responsible for these cardiac anomalies. Such discoveries are essential for enhancing diagnostic screening and refining therapeutic interventions, ultimately improving the lives of individuals affected by these conditions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (© 2024 Soliman, Acharya, Gilard, Duffy, Wijns, Kannan and Sultan.)

A critical cell type participating in cardiac outflow tract development is a subpopulation of the neural crest cells, the cardiac neural crest cells (NCCs), whose defect causes a spectrum of cardiovascular abnormalities. Accumulating evidence indicates that mTOR, which belongs to the PI3K-related kinase family and impacts multiple signaling pathways in a variety of contexts, plays a pivotal role for NCC development. Here, we investigated functional roles of mTOR for cardiac neural crest development using several lines of mouse genetic models. We found that disruption of mTOR caused NCC defects and failure of cardiac outflow tract separation, which resulted in a spectrum of cardiac defects including persistent truncus arteriosus, ventricular septal defect and ventricular wall defect. Specifically, mutant neural crest cells showed reduced migration into the cardiac OFT and prematurely exited the cell cycle. A number of critical factors and fundamental signaling pathways, which are important for neural crest and cardiomyocyte development, were impaired. Moreover, actin dynamics was disrupted by mTOR deletion. Finally, by phenotyping the neural crest Rptor and Rictor knockout mice respectively, we demonstrate that mTOR acts principally through the mTORC1 pathway for cardiac neural crest cells. Altogether, these data established essential roles of mTOR for cardiac NCC development and imply that dysregulation of mTOR in NCCs may underline a spectrum of cardiac defects.

Congenital heart defects (CHDs) affecting the cardiac outflow tract (OFT) constitute a significant cause of morbidity and mortality. The OFT develops from migratory cell populations which include the cardiac neural crest cells (cNCCs) and secondary heart field (SHF) derived myocardium and endocardium. The related transcription factors HAND1 and HAND2 have been implicated in human CHDs involving the OFT. Although Hand1 is expressed within the OFT, Hand1 NCC-specific conditional knockout mice (H1CKOs) are viable. Here we show that these H1CKOs present a low penetrance of OFT phenotypes, whereas SHF-specific Hand1 ablation does not reveal any cardiac phenotypes. Further, HAND1 and HAND2 appear functionally redundant within the cNCCs, as a reduction/ablation of Hand2 on an NCC-specific H1CKO background causes pronounced OFT defects. Double conditional Hand1 and Hand2 NCC knockouts exhibit persistent truncus arteriosus (PTA) with 100% penetrance. NCC lineage-tracing and Sema3c in situ mRNA expression reveal that Sema3c-expressing cells are mis-localized, resulting in a malformed septal bridge within the OFTs of H1CKO;H2CKO embryos. Interestingly, Hand1 and Hand2 also genetically interact within the SHF, as SHF H1CKOs on a heterozygous Hand2 background exhibit Ventricular Septal Defects (VSDs) with incomplete penetrance. Previously, we identified a BMP, HAND2, and GATA-dependent Hand1 OFT enhancer sufficient to drive reporter gene expression within the nascent OFT and aorta. Using these transcription inputs as a probe, we identify a novel Hand2 OFT enhancer, suggesting that a conserved BMP-GATA dependent mechanism transcriptionally regulates both HAND factors. These findings support the hypothesis that HAND factors interpret BMP signaling within the cNCCs to cooperatively coordinate OFT morphogenesis.

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.

Congenital heart disease (CHD) is the most common birth defect, and the leading cause of death due to birth defects, yet causative molecular mechanisms remain mostly unknown. We previously implicated a novel CHD candidate gene, SHROOM3, in a patient with CHD. Using a Shroom3 gene trap knockout mouse (Shroom3(gt/gt)) we demonstrate that SHROOM3 is downstream of the noncanonical Wnt planar cell polarity signaling pathway (PCP) and loss-of-function causes cardiac defects. We demonstrate Shroom3 expression within cardiomyocytes of the ventricles and interventricular septum from E10.5 onward, as well as within cardiac neural crest cells and second heart field cells that populate the cardiac outflow tract. We demonstrate that Shroom3(gt/gt) mice exhibit variable penetrance of a spectrum of CHDs that include ventricular septal defects, double outlet right ventricle, and thin left ventricular myocardium. This CHD spectrum phenocopies what is observed with disrupted PCP. We show that during cardiac development SHROOM3 interacts physically and genetically with, and is downstream of, key PCP signaling component Dishevelled 2. Within Shroom3(gt/gt) hearts we demonstrate disrupted terminal PCP components, actomyosin cytoskeleton, cardiomyocyte polarity, organization, proliferation and morphology. Together, these data demonstrate SHROOM3 functions during cardiac development as an actomyosin cytoskeleton effector downstream of PCP signaling, revealing SHROOM3’s novel role in cardiac development and CHD.

Alcohol consumption is increasing worldwide. Many child-bearing-aged women consume alcohol during pregnancy, intentionally or unintentionally, thereby increasing the potential risk for severe congenital diseases. Congenital heart disease (CHD) is the most common birth defect worldwide and can result from both hereditary and acquired factors. Prenatal alcohol exposure (PAE) is considered a key factor that leads to teratogenesis in CHD and its specific phenotypes, especially defects of the cardiac septa, cardiac valves, cardiac canals, and great arteries, adjacent to the chambers, both in animal experiments and clinical retrospective studies. The mechanisms underlying CHD and its phenotypes caused by PAE are associated with changes in retinoic acid biosynthesis and its signaling pathway, apoptosis and defective function of cardiac neural crest cells, disturbance of the Wntβ-catenin signaling pathway, suppression of bone morphogenetic protein (BMP) signaling, and other epigenetic mechanisms. Drug supplements and early diagnosis can help prevent PAE from inducing CHDs.

Cardiovascular development is a complex developmental process in which multiple cell lineages are involved, namely the deployment of first and second heart fields. Beside the contribution of these cardiogenic fields, extracardiac inputs to the developing heart are provided by the migrating cardiac neural crest cells and the proepicardial derived cells. The proepicardium (PE) is a transitory cauliflower-like structure located between the cardiac and hepatic primordia. The PE is constituted by an internal mesenchymal component surrounded by an external epithelial lining. With development, cells derived from the proepicardium migrate to the neighboring embryonic heart and progressive cover the most external surface, leading to the formation of the embryonic epicardium. Experimental evidence in chicken have nicely demonstrated that epicardial derived cells can distinctly contribute to fibroblasts, endothelial and smooth muscle cells. Surprisingly, isolation of the developing PE anlage and ex vivo culturing spontaneously lead to differentiation into beating cardiomyocytes, a process that is enhanced by Bmp but halted by Fgf administration. In this study we provide a comprehensive characterization of the developmental expression profile of multiple microRNAs during epicardial development in chicken. Subsequently, we identified that miR-125, miR-146, miR-195 and miR-223 selectively enhance cardiomyogenesis both in the PE/ST explants as well as in the embryonic epicardium, a Smurf1- and Foxp1-driven process. In addition we identified three novel long non-coding RNAs with enhanced expression in the PE/ST, that are complementary regulated by Bmp and Fgf administration and well as by microRNAs that selectively promote cardiomyogenesis, supporting a pivotal role of these long non coding RNAs in microRNA-mediated cardiomyogenesis of the PE/ST cells.

Outflow tract abnormalities are the most frequent congenital heart defects. These are due to the absence or dysfunction of the two main cell types, i.e., neural crest cells and secondary heart field cells that migrate in opposite directions at the same stage of development. These cells directly govern aortic arch patterning and development, ascending aorta dilatation, semi-valvular and coronary artery development, aortopulmonary septation abnormalities, persistence of the ductus arteriosus, trunk and proximal pulmonary arteries, sub-valvular conal ventricular septal/rotational defects, and non-compaction of the left ventricle. In some cases, depending on the functional defects of these cells, additional malformations are found in the expected spatial migratory area of the cells, namely in the pharyngeal arch derivatives and cervico-facial structures. Associated non-cardiovascular anomalies are often underestimated, since the multipotency and functional alteration of these cells can result in the modification of multiple neural, epidermal, and cervical structures at different levels. In most cases, patients do not display the full phenotype of abnormalities, but congenital cardiac defects involving the ventricular outflow tract, ascending aorta, aortic arch and supra-aortic trunks should be considered as markers for possible impaired function of these cells. Neural crest cells should not be considered as a unique cell population but on the basis of their cervical rhombomere origins R3-R5 or R6-R7-R8 and specific migration patterns: R3-R4 towards arch II, R5-R6 arch III and R7-R8 arch IV and VI. A better understanding of their development may lead to the discovery of unknown associated abnormalities, thereby enabling potential improvements to be made to the therapeutic approach.

The migratory cardiac neural crest cells (CNCCs) contribute greatly to cardiovascular development. A thorough understanding of the cell lineages, developmental chronology, and transcriptomic states of CNCC derivatives during normal development is essential for deciphering the pathogenesis of CNCC‐associated congenital anomalies. Here, we perform single‐cell transcriptomic sequencing of 34,131 CNCC‐derived cells in mouse hearts covering eight developmental stages between E10.5 and P7. We report the presence of CNCC‐derived mural cells that comprise pericytes and microvascular smooth muscle cells (mVSMCs). Furthermore, we identify the transition from the CNCC‐derived pericytes to mVSMCs and the key regulators over the transition. In addition, our data support that many CNCC derivatives had already committed or differentiated to a specific lineage when migrating into the heart. We explore the spatial distribution of some critical CNCC‐derived subpopulations with single‐molecule fluorescence in situ hybridization. Finally, we computationally reconstruct the differentiation path and regulatory dynamics of CNCC derivatives. Our study provides novel insights into the cell lineages, developmental chronology, and regulatory dynamics of CNCC derivatives during development., A single‐cell transcriptomic atlas of mouse cardiac neural crest cell (CNCC) derivatives reveals that many had already committed or differentiated to a specific lineage when migrating into the heart.

Congenital heart defects (CHDs) represent the most common human birth defects. Our previous study indicates that the malfunction of microRNAs (miRNAs) in cardiac neural crest cells (NCCs), which contribute to the development of the heart and the connected great vessels, is likely linked to the pathogenesis of human CHDs. In this study, we attempt to further search for causative single-nucleotide variants (SNVs) from CHD patients that mediate the mis-regulating of miRNAs on their downstream target genes in the pathogenesis of CHDs. As a result, a total of 2,925 3′UTR SNVs were detected from a CHD cohort. In parallel, we profiled the expression of miRNAs in cardiac NCCs and found 201 expressed miRNAs. A combined analysis with these data further identified three 3′UTR SNVs, including NFATC1 c.*654C>T, FGFRL1 c.*414C>T, and CTNNB1 c.*729_*730insT, which result in the malfunction of miRNA-mediated gene regulation. The dysregulations were further validated experimentally. Therefore, our study indicates that miRNA-mediated gene dysregulation in cardiac NCCs could be an important etiology of congenital heart disease, which could lead to a new direction of diagnostic and therapeutic investigation on congenital heart disease.

Cells display distinct metabolic characteristics depending on its differentiation stage. The fuel type of the cells serves not only as a source of energy but also as a driver of differentiation. Glucose, the primary nutrient to the cells, is a critical regulator of rapidly growing embryos. This metabolic change is a consequence as well as a cause of changes in genetic program. Disturbance of fetal glucose metabolism such as in diabetic pregnancy is associated with congenital heart disease. In utero hyperglycemia impacts the left-right axis establishment, migration of cardiac neural crest cells, conotruncal formation and mesenchymal formation of the cardiac cushion during early embryogenesis and causes cardiac hypertrophy in late fetal stages. In this review, we focus on the role of glucose in cardiogenesis and the molecular mechanisms underlying heart diseases associated with hyperglycemia.

The neural crest (NC) is a multipotent and temporarily migratory cell population stemming from the dorsal neural tube during vertebrate embryogenesis. Cardiac neural crest cells (NCCs), a specified subpopulation of the NC, are vital for normal cardiovascular development, as they significantly contribute to the pharyngeal arch arteries, the developing cardiac outflow tract (OFT), cardiac valves, and interventricular septum. Various signaling pathways are shown to orchestrate the proper migration, compaction, and differentiation of cardiac NCCs during cardiovascular development. Any loss or dysregulation of signaling pathways in cardiac NCCs can lead to abnormal cardiovascular development during embryogenesis, resulting in abnormalities categorized as congenital heart defects (CHDs). This review focuses on the contributions of cardiac NCCs to cardiovascular formation, discusses cardiac defects caused by a disruption of various regulatory factors, and summarizes the role of multiple signaling pathways during embryonic development. A better understanding of the cardiac NC and its vast regulatory network will provide a deeper insight into the mechanisms of the associated abnormalities, leading to potential therapeutic advancements.

In higher vertebrates, cephalic neural crest cells (NCCs) form craniofacial skeleton by differentiating into chondrocytes and osteoblasts. A subpopulation of cephalic NCCs, cardiac NCCs (CNCCs), migrates to the heart. However, CNCCs mostly do not yield skeletogenic derivatives, and the molecular mechanisms of this fate restriction remain elusive. We identify a disintegrin and metalloprotease 19 (Adam19) as a position-specific fate regulator of NCCs. Adam19-depleted mice abnormally form NCC-derived cartilage in their hearts through the upregulation of Sox9 levels in CNCCs. Moreover, NCC-lineage-specific Sox9-overexpressing mice recapitulate CNCC chondrogenesis. In vitro experiments show that Adam19 mediates the cleavage of bone morphogenic protein (BMP) type I receptor Alk2 (Acvr1), whereas pharmacogenetic approaches reveal that Adam19 inhibits CNCC chondrogenesis by suppressing the BMP-Sox9 cascade, presumably through processing Alk2. These findings suggest a metalloprotease-dependent mechanism attenuating cellular responsiveness to BMP ligands, which is essential for both the positional restriction of NCC skeletogenesis and normal heart development., 心臓内での軟骨形成をおさえる仕組みを解明 --プロテアーゼが細胞の軟骨分化を防ぐ--. 京都大学プレスリリース. 2019-10-21.

Background Congenital cardiovascular malformations are the most common birth defects affecting children. Several of these defects occur in structures developing from neural crest cells. One of the key signaling pathways regulating cardiac neural crest cell (CNCC) development involves the endothelin-A receptor (Ednra). However, the exact function of Ednra signaling in CNCC is unknown. Results The fate mapping of CNCC in Ednra embryos indicated that the migration of these cells is aberrant in the cardiac outflow tract (OFT), but not in the pharyngeal arches. This premature arrest of CNCC migration occurs independently of CNCC proliferation and apoptosis changes and major gene expression changes. Analysis of the Rho family of small GTPases in the mutant embryos revealed that Cdc42 failed to localize normally in the CNCC migrating in the OFT. The inhibition of Cdc42 activity in cultured embryos recapitulated the migratory phenotype observed in Ednra mice. Further analyses revealed that Cdc42 is part of the signaling pathway activated by endothelin specifically in OFT CNCC to control their migration. Conclusions These results indicated that the activation of Cdc42 by endothelin signaling is important for CNCC migration in the OFT but this pathway is not involved in mandibular or pharyngeal arch artery patterning.

Background The neural crest is a transient structure present in early embryogenesis. Cephalic neural crest cells migrate into the pharyngeal arches and the frontonasal process that becomes the forehead and midfacial structures. They also contribute to forming the media of the arteries of the circle of Willis and their branches. The cardiac neural crest produces vascular smooth muscle cells in the ascending aorta, cardiac septum and coronary arteries. Methods In this review, we evaluate the role of the neural crest in moyamoya disease and the pathological implications from the concurrence of moyamoya disease and cardiovascular diseases from the point of view of neural crest cell distributions. Results Midline craniofacial and central nervous system anomalies with eye anomalies, morning glory disc anomaly in patients with moyamoya disease can both be explained as a subtype of cephalic neurocristopathy. Further, the association between moyamoya disease and cardiac manifestations (congenital cardiac defects and coronary artery disease) have also been reported. Both the cephalic neural crest and cardiac neural crest contribute to these concurrent arterial diseases, as cardio-cephalic neurocristopathy. Conclusion The concept of cephalic/cardio-cephalic neurocristopathy provides a new perspective to understanding the underlying aetiological associations and to developing future therapeutic approaches for concomitant moyamoya disease and cardiovascular diseases.

The cardiac neural crest cells (cNCCs) is a transient, migratory cell population that contribute to the formation of major arteries and the septa and valves of the heart. Abnormal development of cNCCs leads to a spectrum of congenital heart defects that mainly affect the outflow region of the hearts. Signaling molecules and transcription factors are the best studied regulatory events controlling cNCC development. In recent years, however, accumulated evidence supports that epigenetic regulation also plays an important role in cNCC development. Here, we summarize the functions of epigenetic regulators during cNCC development as well as cNCC related cardiovascular defects. These factors include ATP-dependent chromatin remodeling factors, histone modifiers and DNA methylation modulators. In many cases, mutations in the genes encoding these factors are known to cause inborn heart diseases. A better understanding of epigenetic regulators, their activities and their roles during heart development will ultimately contribute to the development of new clinical applications for patients with congenital heart disease.

Cardiac neural crest cells arise in the caudal hindbrain and then migrate to the heart through the pharyngeal arches. These cells contribute to the formation of the heart, including the outflow tract, and are unique to this neural crest population. MafB is a transcription factor expressed specifically in early migrating cardiac neural crest cells as well as in rhombomeres (r) 5 and 6. Here, we identified the regulatory region in the chicken genome controlling the expression of endogenous MafB transcripts and used these essential elements to express MafB in the cardiac neural crest in reporter assays. A reporter driven by this regulatory region was employed to trace the migration of these cells into the pharyngeal arches. This regulatory region demonstrated transcriptional activity in the cardiac neural crest but not in other neural crest cell subpopulations, such as the cranial and trunk cells. This study provides insights into the gene regulatory mechanisms that specify cardiac neural crest cells among neural crest cell populations.

Cells previously thought to mainly form nerves in the heart turn out to be very important in cardiac muscle formation., The degree to which populations of cardiac progenitors (CPCs) persist in the postnatal heart remains a controversial issue in cardiobiology. To address this question, we conducted a spatiotemporally resolved analysis of CPC deployment dynamics, tracking cells expressing the pan-CPC gene Isl1. Most CPCs undergo programmed silencing during early cardiogenesis through proteasome-mediated and PRC2 (Polycomb group repressive complex 2)–mediated Isl1 repression, selectively in the outflow tract. A notable exception is a domain of cardiac neural crest cells (CNCs) in the inflow tract. These “dorsal CNCs” are regulated through a Wnt/β-catenin/Isl1 feedback loop and generate a limited number of trabecular cardiomyocytes that undergo multiple clonal divisions during compaction, to eventually produce ~10% of the biventricular myocardium. After birth, CNCs continue to generate cardiomyocytes that, however, exhibit diminished clonal amplification dynamics. Thus, although the postnatal heart sustains cardiomyocyte-producing CNCs, their regenerative potential is likely diminished by the loss of trabeculation-like proliferative properties.

Cardiac neural crest cells (cNCCs) are a migratory cell population that stem from the cranial portion of the neural tube. They undergo epithelial-to-mesenchymal transition and migrate through the developing embryo to give rise to portions of the outflow tract, the valves and the arteries of the heart. Recent lineage-tracing experiments in chick and zebrafish embryos have shown that cNCCs can also give rise to mature cardiomyocytes. These cNCC-derived cardiomyocytes appear to be required for the successful repair and regeneration of injured zebrafish hearts. In addition, recent work examining the response to cardiac injury in the mammalian heart has suggested that cNCC-derived cardiomyocytes are involved in the repair/regeneration mechanism. However, the molecular signature of the adult cardiomyocytes involved in this repair is unclear. In this Review, we examine the origin, migration and fates of cNCCs. We also review the contribution of cNCCs to mature cardiomyocytes in fish, chick and mice, as well as their role in the regeneration of the adult heart.

Introduction Congenital anomalies such as ventricular septal defects and truncus communis have been reported with the prenatal use of antiretroviral therapy. The mechanism of antiretroviral therapy teratogenicity is unclear and is therefore the focus of this study. Some human immunodeficiency virus patients on antiretrovirals are placed on antiepileptic drugs which are also teratogenic. The interactive effects arising from this therapeutic combination may affect their teratogenic propensity through their effects on neural crest cell migration. Methods Appropriately cultured neural crest cells from dissected neural tubes of 32-hr old quail embryos exposed to culture media containing peak plasma levels of Atripla, Topiramate and the combination of both were studied. Distance of migration of neural crest cells was measured using the migration assay and the cells were stained with rhodamine phalloidin to evaluate the cell actin. Also quail neural crest cells were brought into suspension and microinjected into chick hosts to determine the migration of the cells to the interventricular septum. Results Migration of cultured neural crest cells was extensive in the control cultures, but inhibited in the treated groups. The experimental cultures showed a disarray of actin cytoskeleton contrary to normal distribution of actin filaments in controls. Significantly, few quail neural crest cells migrated to the interventricular septum of chick host embryos compared to the control cultures. The coadministration of topiramate with antiretroviral therapy does not seem to affect the activity of the antiretroviral drug. Conclusion These results indicate that Atripla and Topiramate cause ventricular septal defects by inhibiting the migration of cardiac neural crest cells.

The recent recovery of mutations in vesicular trafficking genes causing congenital heart disease (CHD) revealed an unexpected role for the endocytic pathway. We now show that mice with a C4232R missense mutation in Low density lipoprotein receptor related protein 1 (LRP1) exhibit atrioventricular septal defects with double outlet right ventricle. Lrp1m/m mice exhibit shortened outflow tracts (OFT) and dysmorphic hypocellular cushions with reduced proliferation and increased apoptosis. Lrp1m/m embryonic fibroblasts show decreased cell motility and focal adhesion turnover associated with retention of mutant LRP1 in endoplasmic reticulum and reduced LRP1 expression. Conditional deletion of Lrp1 in cardiac neural crest cells (CNC) replicates the full CHD phenotype. Cushion explants showed defective cell migration, with gene expression analysis indicating perturbation of Wnt and other signaling pathways. Thus, LRP1 function in CNCs is required for normal OFT development with other cell lineages along the CNC migratory path playing a supporting role., Lin et al. find that mutation in endocytic trafficking protein Lrp1 causes congenital heart defects in mice due to a requirement for Lrp1 in the neural crest lineage, where it regulates outflow tract lengthening. This study provides insights into how Lrp1 and the neural crest contribute to heart development.

Congenital heart defects (CHDs) occur with such a frequency that they constitute a significant cause of morbidity and mortality in both children and adults. A significant portion of CHDs can be attributed to aberrant development of the cardiac outflow tract (OFT), and of one of its cellular progenitors known as the cardiac neural crest cells (NCCs). The gene regulatory networks that identify cardiac NCCs as a distinct NCC population are not completely understood. Heart and neural crest derivatives (HAND) bHLH transcription factors play essential roles in NCC morphogenesis. The Hand1PA/OFT enhancer is dependent upon bone morphogenic protein (BMP) signaling in both cranial and cardiac NCCs. The Hand1PA/OFT enhancer is directly repressed by the endothelin-induced transcription factors DLX5 and DLX6 in cranial but not cardiac NCCs. This transcriptional distinction offers the unique opportunity to interrogate NCC specification, and to understand why, despite similarities, cranial NCC fate determination is so diverse. We generated a conditionally active transgene that can ectopically express DLX5 within the developing mouse embryo in a Cre-recombinase-dependent manner. Ectopic DLX5 expression represses cranial NCC Hand1PA/OFT-lacZ reporter expression more effectively than cardiac NCC reporter expression. Ectopic DLX5 expression induces broad domains of NCC cell death within the cranial pharyngeal arches, but minimal cell death in cardiac NCC populations. This study shows that transcription control of NCC gene regulatory programs is influenced by their initial specification at the dorsal neural tube.

Cardiac neural crest cells (cNCCs) are required for normal heart development. cNCCs are a multipotent and migratory cell lineage that differentiates into multiple cell types. cNCCs migrate into the developing heart to contribute to the septation of the cardiac outflow tract (OFT). Foxc1 and Foxc2 are closely related members of the FOX (Forkhead box) transcription factor family and are expressed in cNCC during heart development. However, the precise role of Foxc1 and Foxc2 in cNCCs has yet to be fully described. We found that compound NCC-specific Foxc1;Foxc2 mutant embryos exhibited persistent truncus arteriosus (PTA), ventricular septal defects (VSDs), and thinning of the ventricular myocardium. Loss of Foxc1/c2 expression in cNCCs resulted in abnormal patterns of cNCC migration into the OFT without the formation of the aorticopulmonary septum. Further, loss of Foxc1 expression in cNCCs resulted in normal OFT development but abnormal ventricular septal formation. In contrast, loss of Foxc2 expression in NCCs led to no obvious cardiac abnormalities. Together, we provide evidence that Foxc1 and Foxc2 in cNCCs are cooperatively required for proper cNCC migration, the formation of the OFT septation, and the development of the ventricles. Our data also suggests that Foxc1 expression may play a larger role in ventricular development compared to Foxc2.

The two cardiac progenitor cell lineages, cardiac neural crest cells (cNCCs) and the second heart field (SHF), play key roles in the development of the cardiac outflow tract (OFT). Both cardiac progenitor cells interact with each other and contribute to OFT formation cooperatively. The neurovascular guiding molecule, semaphorin 3c (Sema3c), is thought to serve as a key attractant for the migration of cNCCs. A previous study reported that Tbx1 null mice showed a significant reduction in Sema3c expression in the OFT region [1]. However, the regulatory effect of Tbx1 on Sema3c was unclear. Here, we show that Sema3c plays key roles in cNCCs-SHF interactions through the regulation by Tbx1 and other molecules during OFT development [2].

Congenital central hypoventilation syndrome (CCHS, OMIM 209880) is a rare autosomal dominant disorder caused by mutation in PHOX2B that manifests as a consequence of abnormal neural crest cell migration during embryogenesis. Unlike other neurocristopathies, however, its impact on the cardiovascular system has not been previously assessed. This study was an effort to characterize the association between congenital heart disease (CHD) and mutations in PHOX2B in patients with CCHS. A retrospective review of patients with CCHS in conjunction with functional analysis of PHOX2B mutations associated with CHD was performed. To substantiate functional implications of identified variants, we conducted protein structure analyses and in silico mutagenesis were conducted. The prevalence of CHD among patients with CCHS was significantly greater (30%; p

BACKGROUND Proper development of the great vessels of the heart and septation of the cardiac outflow tract requires cardiac neural crest cells. These cells give rise to the parasympathetic cardiac ganglia, the smooth muscle layer of the great vessels, some cardiomyocytes, and the conotruncal cushions and aorticopulmonary septum of the outflow tract. Ablation of cardiac neural crest cells results in defective patterning of each of these structures. Previous studies have shown that targeted deletion of the forkhead transcription factor C2 (Foxc2), results in cardiac phenotypes similar to that derived from cardiac neural crest cell ablation. RESULTS We report that Foxc2-/- embryos on the 129s6/SvEv inbred genetic background display persistent truncus arteriosus and hypoplastic ventricles before embryonic lethality. Foxc2 loss-of-function resulted in perturbed cardiac neural crest cell migration and their reduced contribution to the outflow tract as evidenced by lineage tracing analyses together with perturbed expression of the neural crest cell markers Sox10 and Crabp1. Foxc2 loss-of-function also resulted in alterations in PlexinD1, Twist1, PECAM1, and Hand1/2 expression in association with vascular and ventricular defects. CONCLUSIONS Our data indicate Foxc2 is required for proper migration of cardiac neural crest cells, septation of the outflow tract, and development of the ventricles. Developmental Dynamics 247:1286-1296, 2018. © 2018 Wiley Periodicals, Inc.

Endocytic trafficking is an under-appreciated pathway in cardiac development. Several genes related to endocytic trafficking have been uncovered in a mutagenic ENU screen, in which mutations led to congenital heart defects (CHDs). In this article, we review the relationship between these genes (including LRP1 and LRP2) and cardiac neural crest cells (CNCCs) during cardiac development. Mice with an ENU-induced Lrp1 mutation exhibit a spectrum of CHDs. Conditional deletion using a floxed Lrp1 allele with different Cre drivers showed that targeting neural crest cells with Wnt1-Cre expression replicated the full cardiac phenotypes of the ENU-induced Lrp1 mutation. In addition, LRP1 function in CNCCs is required for normal OFT lengthening and survival/expansion of the cushion mesenchyme, with other cell lineages along the NCC migratory path playing an additional role. Mice with an ENU-induced and targeted Lrp2 mutation demonstrated the cardiac phenotype of common arterial trunk (CAT). Although there is no impact on CNCCs in Lrp2 mutants, the loss of LRP2 results in the depletion of sonic hedgehog (SHH)-dependent cells in the second heart field. SHH is known to be crucial for CNCC survival and proliferation, which suggests LRP2 has a non-autonomous role in CNCCs. In this article, other endocytic trafficking proteins that are associated with CHDs that may play roles in the NCC pathway during development, such as AP1B1, AP2B1, FUZ, MYH10, and HECTD1, are reviewed.

Bicuspid Aortic valve (BAV) is one of the most common congenital cardiac malformations with a prevalence of 1-2% in the general population. Patients with BAV have a 9-fold increased risk of developing serious secondary complications including stenosis, endocarditis, regurgitation, dilation of the aorta, aortic aneurysms and subsequent dissection resulting in a significant increase in morbidity. Progressive decline in valve functionality and associated complications warrants surgical intervention in 27% of the affected individuals. The understanding of genetic and molecular mechanisms underlying disease pathology has been largely confounded by phenotypic heterogeneity, incomplete penetrance and variable expressivity. Additionally, the complex interplay between genetic, epigenetic and haemodynamic factors during and after development along with their dynamic expression depending on tissue type contribute to the elusiveness of the disease. While the exact mechanism of pathogenesis remains unclear, recent advances in genetics, propelled by large scale candidate gene discovery strategies employing next generation sequencing, epigenetics, haemodynamic modelling and imaging have provided insights into the development of BAV and associated aortopathy, thus accelerating advances in clinical management and diagnosis of the disease. This review aims at providing a comprehensive understanding of cardiac valve development and the underlying genetic and molecular mechanisms contributing to BAV associated aortopathy.

Normal aortic valves are composed of valve endothelial cells (VECs) and valve interstitial cells (VICs). VICs are the major cell population and have distinct embryonic origins in the endocardium and cardiac neural crest cells. Cell signaling between the VECs and VICs plays critical roles in aortic valve morphogenesis. Disruption of major cell signaling pathways results in aortic valve malformations, including bicuspid aortic valve (BAV). BAV is a common congenital heart valve disease that may lead to calcific aortic valve disease (CAVD), but there is currently no effective medical treatment for this beyond surgical replacement. Mouse and human studies have identified causative gene mutations for BAV and CAVD via disrupted VEC to VIC signaling. Future studies on the developmental signaling mechanisms underlying aortic valve malformations and the pathogenesis of CAVD using genetically modified mouse models and patient-induced pluripotent stem cells may identify new effective therapeutic targets for the disease.

Foxc2, a member of the winged helix transcription factor family, is essential for eye, calvarial bone, cardiovascular and kidney development in mice. Nevertheless, how Foxc2-expressing cells and their descendent cells contribute to the development of these tissues and organs has not been elucidated. Here, we generated a Foxc2 knock-in (Foxc2CreERT2 ) mouse, in which administration of estrogen receptor antagonist tamoxifen induces nuclear translocation of Cre recombinase in Foxc2-expressing cells. By crossing with ROSA-LacZ reporter mice (Foxc2CreERT2 ; R26R), the fate of Foxc2 positive (Foxc2+ ) cells was analyzed through LacZ staining at various embryonic stages. We found Foxc2+ cell descendants in the supraoccipital and exoccipital bone in E18.5 embryos, when tamoxifen was administered at embryonic day (E) 8.5. Furthermore, Foxc2+ descendant cranial neural crest cells at E8-10 were restricted to the corneal mesenchyme, while Foxc2+ cell derived cardiac neural crest cells at E6-12 were found in the aorta, pulmonary trunk and valves, and endocardial cushions. Foxc2+ cell descendant contributions to the glomerular podocytes in the kidney were also observed following E6.5 tamoxifen treatment. Our results are consistent with previous reports of Foxc2 expression during early embryogenesis and the Foxc2CreERT2 mouse provides a tool to investigate spatiotemporal roles of Foxc2 and contributions of Foxc2+ expressing cells during mouse embryogenesis.

Armadillo repeat and Armadillo-like helical domain containing proteins form a large family with diverse and fundamental functions in many eukaryotes. Herein we investigated the spatiotemporal expression pattern of Armadillo-like helical domain containing 4 (or Armh4) as an uncharacterized protein coding mouse gene, within the mouse embryo during the initial stages of heart morphogenesis. We found Armh4 is initially expressed in both first heart field as well as the second heart field progenitors and subsequently within predominantly their cardiomyocyte derivatives. Armh4 expression is initially cardiac-restricted in the developing embryo and is expressed in second heart field subpharyngeal mesoderm prior to cardiomyocyte differentiation, but Armh4 diminishes as the embryonic heart matures into the fetal heart. Armh4 is subsequently expressed in craniofacial structures and neural crest-derived dorsal root and trigeminal ganglia. Whereas lithium chloride-induced stimulation of Wnt/β-catenin signaling elevated Armh4 expression in both second heart field subpharyngeal mesodermal progenitors and outflow tract, right ventricle and atrial cardiomyocytes, neither a systemic loss of Islet-1 nor an absence of cardiac neural crest cells had any effect upon Armh4 expression. These results confirm that Wnt/β-catenin-responsive Armh4 is a useful specific biomarker of the FHF and SHF cardiomyocyte derivatives only.

Conotruncal heart defects (CTDs) are closely related to defective outflow tract (OFT) development, in which cardiac neural crest cells (CNCCs) play an indispensable role. However, the genetic etiology of CTDs remains unclear. Mesoderm posterior 2 (MESP2) is an important transcription factor regulating early cardiogenesis. Nevertheless, MESP2 variants have not been reported in congenital heart defect (CHD) patients. We first identified four MESP2 variants in 601 sporadic nonsyndromic CTD patients that were not detected in 400 healthy controls using targeted sequencing. Reverse transcription-quantitative PCR (RT-qPCR), immunohistochemistry, and immunofluorescence assays revealed MESP2 expression in the OFT of Carnegie stage (CS) 11, CS13, and CS15 human embryos and embryonic day (E) 8.5, E10, and E11.5 mouse embryos. Functional analyses in HEK 293T cells, HL-1 cells, JoMa1 cells, and primary mouse CNCCs revealed that MESP2 directly regulates the transcriptional activities of downstream CTD-related genes and promotes CNCC proliferation by regulating cell cycle factors. Three MESP2 variants, c.346G>C (p.G116R), c.921C>G (p.Y307X), and c.59A>T (p.Q20L), altered the transcriptional activities of MYOCD, GATA4, NKX2.5, and CFC1 and inhibited CNCC proliferation by upregulating p21cip1 or downregulating Cdk4. Based on our findings, MESP2 variants disrupted MESP2 function by interfering with CNCC proliferation during OFT development, which may contribute to CTDs. KEY MESSAGES: This study first analyzed MESP2 variants identified in sporadic nonsyndromic CTD patients. MESP2 is expressed in the OFT of different stages of human and mouse embryos. MESP2 regulates the transcriptional activities of downstream CTD-related genes and promotes CNCC proliferation by regulating cell cycle factor p21cip1 or Cdk4.

RationaleCardiac neural crest cells (CNCCs) contribute greatly to cardiovascular development. A thorough understanding of the cell lineages, transcriptomic states and regulatory networks of CNCC derivatives during normal development is essential for deciphering the pathogenesis of CNCC-associated congenital anomalies. However, the transcriptomic landscape of CNCC derivatives during development has not yet been examined at a single-cell resolution.ObjectiveWe sought to systematically characterize the cell lineages, define the developmental chronology and elucidate the transcriptomic dynamics of CNCC derivatives during embryonic and neonatal development.Methods and ResultsWe performed single-cell transcriptomic sequencing of 34,131 CNCC-derived cells in mouse hearts from eight developmental stages between E10.5 and P7. Through single-cell analyses and single-molecule fluorescencein situhybridization, we confirmed the presence of CNCC-derived mural cells. Furthermore, we found the transition from CNCC-derived pericytes to microvascular smooth muscle cells, and identified the genes that were significantly regulated during this transition through pseudo-temporal analysis. CNCC-derived neurons first appeared at E10.5, which was earlier than previously recognized. In addition, the CNCC derivatives switched from a proliferative to a quiescent state with the progression of development. Gradual loss of the neural crest molecular signature with development was also observed in the CNCC derivatives. Our data suggested that many CNCC-derivatives had already committed or differentiated to a specific lineage when migrating to the heart. Finally, we characterized some previously unknown subpopulations of CNCC derivatives during development. For example, we found thatPenk+ cells, which were mainly localized in outflow tract cushions, were all derived from CNCCs.ConclusionsOur study provides novel insights into the cell lineages, molecular signatures, developmental chronology and state change dynamics of CNCC derivatives during embryonic and neonatal development. Our dataset constitutes a valuable resource that will facilitate future efforts in exploring the role of CNCC derivatives in development and disease.

Cardiac neural crest cells contribute to important portions of the cardiovascular system including the aorticopulmonary septum and cardiac ganglion. Using replication incompetent avian retroviruses for precise high-resolution lineage analysis, we uncover a previously undescribed neural crest contribution to cardiomyocytes of the ventricles in Gallus gallus, supported by Wnt1-Cre lineage analysis in Mus musculus. To test the intriguing possibility that neural crest cells contribute to heart repair, we examined Danio rerio adult heart regeneration in the neural crest transgenic line, Tg(−4.9sox10:eGFP). Whereas the adult heart has few sox10+ cells in the apex, sox10 and other neural crest regulatory network genes are upregulated in the regenerating myocardium after resection. The results suggest that neural crest cells contribute to many cardiovascular structures including cardiomyocytes across vertebrates and to the regenerating heart of teleost fish. Thus, understanding molecular mechanisms that control the normal development of the neural crest into cardiomyocytes and reactivation of the neural crest program upon regeneration may open potential therapeutic approaches to repair heart damage in amniotes., eLife digest Before birth, unspecialized stem cells go through a process called differentiation to form the many types of cells found in the adult. Neural crest cells are a group of these stem cells found in all animals with backbones (i.e. vertebrates) including humans. These cells migrate extensively during development to form many different parts of the body. Due to their contributions to diverse organs and tissues, neural crest cells are very important for healthy development. The heart ventricle is one of the tissues to which neural crest cells contribute during embryonic development in fish and amphibians. However, it was unclear whether this is also the case for birds or mammals or whether neural crest cells have any roles in the regeneration of the adult heart after injury in fish and amphibians. To address these questions, Tang, Martik et al. used cell biology techniques to track neural crest cells in living animals. The experiments revealed that neural crest cells contribute to heart tissue in developing birds and mammals and help repair the heart in adult zebrafish. Further results showed that the contribution of neural crest cells to the heart is controlled by the same genes during both the growth of the embryonic heart and the repair of the adult heart. These results provide new insights into the repair and healing of damaged heart muscle in fish. They also show that similar processes could exist in mammals, including humans, suggesting that activating neural crest cells in the heart could treat damage caused by heart attacks and related conditions.

SummaryEstablishment of separated pulmonary and systemic circulations in vertebrates relies on the key role of neural crest cells (NCC) for the septation of the embryonic cardiac outflow tract (OFT). Absence of NCCs induces OFT septation defects, analogous to a loss of Bone Morphogenetic Proteins (BMPs) activity, though it remains unclear how BMPs control cardiac NCC differentiation and behaviour. To address this question, we monitored cardiac NCC state upon gain in BMP signaling, caused by the deletion ofDullard, using 3D-imaging and single cell transcriptomics. Specific loss ofDullardin the NCC results in premature OFT septation, pulmonary artery obstruction and embryonic death. This is caused by uncontrolled NCC convergence towards the endocardium and asymmetrical myocardial differentiation, promoted by elevated levels of the guiding cueSema3cand decreased levels in mesenchymal trait markers. Furthermore, we unraveled the molecular basis of the zipper-like OFT septation where graded Sema3c expression follow a gradient of BMP activation in NCC along the OFT length.

The cardiac neural crest originates in the caudal hindbrain, migrates to the heart, and contributes to septation of the cardiac outflow tract and ventricles, an ability unique to this neural crest subpopulation. Here we have used a FoxD3 neural crest enhancer to isolate a pure population of cardiac neural crest cells for transcriptome analysis. This has led to the identification of transcription factors, signaling receptors/ligands, and cell adhesion molecules upregulated in the early migrating cardiac neural crest. We then functionally tested the role of one of the upregulated transcription factors, MafB, and found that it acts as a regulator of Sox10 expression specifically in the cardiac neural crest. Our results not only reveal the genome-wide profile of early migrating cardiac neural crest cells, but also provide molecular insight into what makes the cardiac neural crest unique.