Cardiac Neural Crest Research Articles

Molecular Characterization of Embryonic Stem Cell-Derived Cardiac Neural Crest-Like Cells Revealed a Spatiotemporal Expression of an Mlc-3 Isoform.

Background and ObjectivesPluripotent embryonic stem (ES) cells represent a perfect model system for the investigation of early developmental processes. Besides their differentiation into derivatives of the three primary germ layers, they can also be differentiated into derivatives of the ‘fourth’ germ layer, the neural crest (NC). Due to its multipotency, extensive migration and outstanding capacity to generate a remarkable number of different cell types, the NC plays a key role in early developmental processes. Cardiac neural crest (CNC) cells are a subpopulation of the NC, which are of crucial importance for precise cardiovascular and pharyngeal glands’ development. CNC-associated malformations are rare, but always severe and life-threatening. Appropriate cell models could help to unravel underlying pathomechanisms and to develop new therapeutic options for relevant heart malformations.MethodsMurine ES cells were differentiated according to a mesodermal-lineage promoting protocol. Expression profiles of ES cell-derived progeny at various differentiation stages were investigated on transcript and protein level.ResultsComparative expression profiling of murine ES cell multilineage progeny versus undifferentiated ES cells confirmed differentiation into known cell derivatives of the three primary germ layers and provided evidence that ES cells have the capacity to differentiate into NC/CNC-like cells. Applying the NC/CNC cell-specific marker, 4E9R, an unambiguous identification of ES cell-derived NC/CNC-like cells was achieved.ConclusionsOur findings will facilitate the establishment of an ES cell-derived CNC cell model for the investigation of molecular pathways during cardiac development in health and disease.

Krox20 Regulates Endothelial Nitric Oxide Signaling in Aortic Valve Development and Disease.

Among the aortic valve diseases, the bicuspid aortic valve (BAV) occurs when the aortic valve has two leaflets (cusps), rather than three, and represents the most common form of congenital cardiac malformation, affecting 1–2% of the population. Despite recent advances, the etiology of BAV is poorly understood. We have recently shown that Krox20 is expressed in endothelial and cardiac neural crest derivatives that normally contribute to aortic valve development and that lack of Krox20 in these cells leads to aortic valve defects including partially penetrant BAV formation. Dysregulated expression of endothelial nitric oxide synthase (Nos3) is associated with BAV. To investigate the relationship between Krox20 and Nos3 during aortic valve development, we performed inter-genetic cross. While single heterozygous mice had normal valve formation, the compound Krox20+/−;Nos3+/− mice had BAV malformations displaying an in vivo genetic interaction between these genes for normal valve morphogenesis. Moreover, in vivo and in vitro experiments demonstrate that Krox20 directly binds to Nos3 proximal promoter to activate its expression. Our data suggests that Krox20 is a regulator of nitric oxide in endothelial-derived cells in the development of the aortic valve and concludes on the interaction of Krox20 and Nos3 in BAV formation.

Cardiac myxoma and neural crests: a tense relationship.

In cardiac myxomas, the malignant transformation process, selecting incidental gene mutations and leading to loss of proliferation control, has not a so drastic effects in terms of growth rate of tumor mass, but frequently the particular location of lesion engrosses the high risk for health. For accurate cancer cell profiling, it is important to establish the embryologic origin of malignant cells and their initial commitments, above all, in the sight of therapeutic strategies and solutions. Here, we advance, for cardiac myxoma, the hypothesis of an origin from cardiac neural crestcells and we attempt to support it by an integrated discussion of current knowledge about embryological characteristics of neural crestcells and most recent studies focusing cardiac myxomas. We discuss the relationship between the basic plasticity of cardiac neural crestcells and some typical mutations arising in neoplastic lesions as well as the expression of typical cell markers of neural crests derivatives. Dysfunctions in proliferative and migratory programs, focused in other studies, are evaluated in the context of the topological and histopathological characteristics of cardiac myxomas.

Armadillo-like helical domain containing-4 is dynamically expressed in both the first and second heart fields

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.

Metalloprotease-Dependent Attenuation of BMP Signaling Restricts Cardiac Neural Crest Cell Fate

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. Invitro 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.

Cardiac neural crest contributes to cardiomyocytes in amniotes and heart regeneration in zebrafish.

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.

Abstract 627: Primary Cilia of the Cardiac Neural Crest Orchestrate Critical Aspects of Ventricular Maturation and Postnatal Cardiac Function: A Role for Hedgehog Signaling

Primary cilia are tiny, plasma-membrane bound organelles that function to modulate intra- and extracellular signaling, in particular Hedgehog signaling, implicated in both normal development as well as in various disease pathologies, including congenital heart disease (CHD). Cardiac neural crest cells (CNCC) display primary cilia and function as a major progenitor cell population contributing to the developing heart. We hypothesized that damage to or loss of primary cilia in CNCC would impair normal heart development, leading to CHD. We present 1) a precise description of CHD’s resulting from loss of primary cilia of CNCC in vivo ; 2) characterization of the functional, electrophysiological aspects of the perinatal phenotype; and 3) a preliminary investigation of the molecular mechanisms leading to this phenotype. Using a Wnt1:Cre-2, Ift88-targeted conditional elimination of primary cilia, and a Td-tomato reporter to track CNCC, we observed loss of cilia in CNCC at embryonic day E9.5, with notable CNCC contribution to the ventricular myocardium and pronounced disorganization of the endocardium by E10.5.The phenotype resulting from cilia loss in CNCC was characterized by a variety predicted CHDs in addition to disorganization of the ventricular endocardium, pronounced noncompaction of the ventricular myocardium, and perinatal lethality. To investigate potential changes in Hedgehog signaling resulting from cilia loss, we implemented an in vivo conditional knockout in which Wnt1:Cre-2 drives expression of a mutant, constitutively-active Hedgehog receptor. Noncompaction observed in all Wnt1:Cre-2/Ift88 mutants became exacerbated in a primary-cilia dependent fashion with the introduction of forced Hedgehog signaling in CNCC. These results support a critical role for both primary cilia and Hedgehog signaling in the development of the ventricular myocardium. Further electrophysiological assessments of Wnt1:Cre-2/Ift88 newborn mutant mice revealed bradycardia, conduction defects, and ECG tracings consistent with ventricular hypertrophy. These data collectively support a role for Hedgehog signaling via primary cilia of CNCC in the pathogenesis of outflow tract defects, VSD, and most notably, ventricular maturation CHDs.

Partially Penetrant Cardiac Neural Crest Defects in Hand1 Phosphomutant Mice: Dimer Choice That Is Not So Critical.

Hand1 is a basic Helix-loop-Helix transcription factor that exhibits post-translationally regulated dimer partner choice that allows for a diverse set of Hand1 transcriptional complexes. Indeed, when Hand1 phosphoregulation is altered, conditionally activated hypophorylation (Hand1PO4-) and phosphorylation mimic (Hand1PO4+) Hand1 alleles disrupt both craniofacial and limb morphogenesis with 100% penetrance. Interestingly, activation of conditional Hand1 Phosphomutant alleles within post-migratory neural crest cells produce heart defects that include ventricular septal defects, double-outlet right ventricle, persistent truncus arteriosus with partial penetrance. Single versus double-lobed thymus is a distinguishing feature between Wnt1-Cre;Hand1PO4-/+ and Wnt1-Cre;Hand1PO4+/+ mice. These data show that although Hand1 dimer regulation plays critical and consistent roles in disrupting craniofacial and limb morphogenesis, Hand1 dimer regulation during cardiac outflow track formation is less critical for normal morphogenesis. This review will present the OFT phenotypes observed in Hand1 Phosphomutant mice, and discuss possible mechanisms of how penetrance differences within the same tissues within the same embryos could be variable.

Cdc42 activation by endothelin regulates neural crest cell migration in the cardiac outflow tract.

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. 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. 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.

Day 0–28 of Human Embryonic Development: Importance of Studying Cell and Tissue Interactions in Head and Heart Development

All vertebrates start their development from a zygote and the processes from this cell to a complex organism are often complicated and include many gene regulatory networks, cell migrations, cell differentiations, communication between cells and between tissues, etc. Earliest mis‐regulations in those processes lead almost always to the demise of the developing embryo – often before the women even realizes she is pregnant. This is because the most exciting and complex processes related to body plan formation happen during the first 8 weeks of embryonic development. Even more fascinating is that at the end of the first months (28 days after fertilization) the head and heart region are clearly distinguishable: the heart is beating, u‐shaped and atria and ventricles are aligned, the pharyngeal arches are present, as are eye and ear anlagen (placodes), in the brain several regions are distinct, including forebrain, midbrain, and hindbrain. There are many remarkable research groups all over the world studying one or the other system (brain, cardiovascular, craniofacial), but only few groups study the interactions between all tissues (ectoderm, mesoderm, endoderm – neural crest as special tissue derived from ectoderm). However, understanding precisely those interactions during earliest and later development will help us to understand human syndromes with the same underlying genetic defect but that present with a huge variety of phenotypes.For example, the DiGeorge Syndrome is caused by a deletion of a region at 22q11.2 that contains 30–40 genes. Due to its involvement of several system it is also called CATCH22 – Cardiac anomalies, Abnormal facies, Thymic hypoplasia, Cleft palate, Hypocalcemia. Each of those abnormalities could be caused by the involvement of cranial neural crest cells during embryonic development, even the heart anomalies as cardiac neural crest cells are part of the posterior population of cranial neural crest cells. Also, in the region of the 22q11.2 is the Tbx1 gene that was shown to be essential during earliest cardiopharyngeal mesoderm development and differentiation. The cardiopharyngeal field is a progenitor field that gives rise to the first heart field and the pharyngeal mesoderm. The latter will differentiate into the second heart field derived cardiac musculature and the branchiomeric (head) muscles. Yet, neither our knowledge of neural crest cell involvement nor our understanding of cardiopharyngeal mesoderm development, fully explains the variety of symptoms seen in DiGeorge Syndrome patients.Therefore, a detailed summary of what is currently known about the first 28 days of head and heart development and the tissue interactions will help us to understand complex syndromes. Furthermore, it will be possible to identify regions that need more research focus. Last, but not least, a better understanding of our own developmental processes also provides insights into evolutionary processes and studying animal developmental processes further contributes to a deepened knowledge of the evolutionary development of our own body. Here, I will present a summary of the first 28 days of head and heart embryonic development in humans and highlight regions of research that next generation scientists might be interested in.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A zebrafish FASD model for congenital heart defects

Mothers who drink during pregnancy expose their developing babies to ethanol, and excessive drinking often produces devastating birth defects collectively termed fetal alcohol spectrum disorder (FASD). These defects include craniofacial defects, cognitive impairment, sensorimotor disabilities and organ deformities, including various congenital heart defects (CHDs), particularly septal and conotruncal defects. Genesis mechanisms for FASD‐associated CHDs are not well understood. Using a zebrafish model, experiments tested whether alcohol interferes with different heart development events or the multiple cardiac precursors that may contribute to CHD genesis. These experiments showed that ethanol affects various cardiac regulatory networks in multiple steps of cardiogenesis (specification, myocardial migration, differentiation, looping, chamber morphogenesis, and endocardial cushion formation). Retinoic acid and folic acid co‐treatment with ethanol had differential rescue effects on FASD‐induced cardiac defects. Early (first heart field) and late‐added (second heart field and cardiac neural crest precursors) cardiac precursors were reduced by ethanol exposure. Experiments showed dysregulation of BMP, Notch and other signaling pathways during atrioventricular valve formation produced persistent valve defects. Together, these results show that embryonic ethanol exposure disrupts critical cardiac morphogenic events and reduce cardiac precursor contributions to the growing heart. Additional mechanistic studies are needed to identify key events that can be targeted therapeutically.Support or Funding InformationNIH/NIAAA AA0022396 and AA026711This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Foxc2 is required for proper cardiac neural crest cell migration, outflow tract septation, and ventricle expansion.

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. 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. 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.

Issue Information

COVER PHOTOGRAPH: Embryo snowflake: Replicated images arranged into the shape of a snowflake, of a coronal section through the pharyngeal arches of an E10.5 Wnt1‐Cre;ROSA‐eYGFP mouse embryo in which neural crest cells (green) and nuclei (blue) are labeled. Image courtesy of Kimberly Inman and Paul Trainor. From: Foxc2 is required for proper cardiac neural crest cell migration, outflow tract septation, and ventricle expansion; Kimberly E. Inman, Carlo Donato Caiaffa, Kristin R. Melton, Lisa L. Sandell, Annita Achilleos, Tsutomu Kume and Paul A. Trainor; Developmental Dynamics 247: pp. 1286–1296.

Abstract 16895: Lrp1, an Endocytic Vesicle Trafficking Protein, is Associated With Congenital Heart Diseases - Through a "non-Autonomous" Action

Backgrounds: Congenital heart diseases affect nearly 1% of neonates per year in the United States and are the leading cause of neonatal death. Despite a strong indication of genetic contribution to congenital heart diseases, the developmental processes and genetic components are poorly understood. From the large-scale forward genetic screen using inbred mice chemically mutagenized with ethyl-nitrosourea (ENU), a novel finding of endocytic trafficking gene mutations, including Lrp1, were recovered as causing congenital heart disease phenotypes. Endocytosis plays roles in modulating cell signaling by regulating the trafficking of the internalized endocytic vesicles to different endosomal compartments, some destined for fusion with lysosomes and degradation while others are recycled to the surface by recycling endosomes. Mutations in these genes causes a spectrum of congenital heart disease comprising of outflow tract abnormalities and alignment defects. Methods and Results: The LRP1 [line 1554(MGI 96828)] mutant mouse line recovered from a forward genetic screen results from a missense (C4232R) mutation in the region encoding the epidermal growth factor (EGF) repeat domain of LRP1. The mutants exhibit a spectrum of septation and outflow tract defects including endocardial cushion defects, double outlet of right ventricle with normal related great arteries as well as DORV with D-malposition of great arteries (Taussig-Bing anomalies). Mouse embryonic fibroblasts (MEF) from LRP1 m/m mutants demonstrated decreased cell-surface expression and increased retention in the endosomes. Migration assay of MEF demonstrated decreased migration of LRP1 m/m compared with control with normal Golgi orientation. The decreasing migration LRP1 m/m also observed in the endocardial cushion tissue from E9.5-10.5 embryos. Different cell lineages crucial to cardiac development with tissue-specific genetic manipulations were used to define the roles of the LRP1 pathway in these three fields of cells during outflow tract development and septation. Cardiac neural crest cell (Wnt1-Cre) knockout of LRP1 recapitulate LRP1 m/m cardiac phenotypes as well as Nkx2.5/Tie2-cre double knockout of LRP1. RNA expression profile suggests Notch pathway and integrin pathway are associated with LRP1 during cardiac development. Conclusions: We provide the first evidence of endocytic vesicle trafficking pathway associated with the development of congenital heart diseases with malalignment/septation and outflow tract defects

Loss of Tbx3 in murine neural crest reduces enteric glia and causes cleft palate, but does not influence heart development or bowel transit

Transcription factors that coordinate migration, differentiation or proliferation of enteric nervous system (ENS) precursors are not well defined. To identify novel transcriptional regulators of ENS development, we performed microarray analysis at embryonic day (E) 17.5 and identified many genes that were enriched in the ENS compared to other bowel cells. We decided to investigate the T-box transcription factor Tbx3, which is prominently expressed in developing and mature ENS. Haploinsufficiency for TBX3 causes ulnar-mammary syndrome (UMS) in humans, a multi-organ system disorder. TBX3 also regulates several genes known to be important for ENS development. To test the hypothesis that Tbx3 is important for ENS development or function, we inactivated Tbx3 in all neural crest derivatives, including ENS progenitors using Wnt1-Cre and a floxed Tbx3 allele. Tbx3 fl/fl; Wnt1-Cre conditional mutant mice die shortly after birth with cleft palate and difficulty feeding. The ENS of mutants was well-organized with a normal density of enteric neurons and nerve fiber bundles, but small bowel glial cell density was reduced. Despite this, bowel motility appeared normal. Furthermore, although Tbx3 is expressed in cardiac neural crest, Tbx3 fl/fl; Wnt1-Cre mice had structurally normal hearts. Thus, loss of Tbx3 within neural crest has selective effects on Tbx3-expressing neural crest derivatives.

Transcriptome profiling of the cardiac neural crest reveals a critical role for MafB

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.

Corneal endothelial cell density and cardiovascular mortality

Based on embryological commonalities between eye and heart development, a global, country-specific meta-analysis of normal, adult corneal endothelial cell density (ECD) was performed and correlated against mortality rates secondary to diseases affecting cardiac neural crest cell (CNCC)-derived cardiovascular structures. A country-specific survey of ECD was performed by searching PubMed for studies reporting ECD datasets from normal adults. All eligible datasets were assigned a country of origin. Country-specific weighted mean ECD were calculated based on dataset n. Country-specific disease mortality rates were obtained from the World Health Organization. The correlations between weighted mean ECD and mortality rates secondary to diseases affecting CNCC-derived cardiovascular structures were calculated. As controls, correlations between ECD and noncardiovascular disease mortality were examined. Pearson correlation coefficients (r) corresponding to P-value < 0.05 were considered significant. Three hundred ninety-two datasets (39,762 eyes) from 267 source-studies were assigned to 42 countries. Significant correlations were found between ECD and mortality due to coronary heart disease (r = -0.39, P = 0.011), hypertension (r = -0.33, P = 0.033), and all-cause cardiac disease (r = -0.36, P = 0.019). No significant correlations were found between ECD and mortality secondary to the control conditions: inflammatory heart disease (mesoderm-derived tissues) (r = -0.12, P = 0.45), diabetes (r = -0.13, P = 0.41), lung disease (r = -0.21, P = 0.18), liver disease (r = -0.13, P = 0.41), renal disease (r = -0.10, P = 0.53), lung cancer (r = 0.02, P = 0.90), pancreatic cancer (r = 0.24, P = 0.13), malnutrition (r = -0.07, P = 0.66), or all-cause mortality (r = 0.04, P = 0.81). Negative correlations exist between ECD and mortality due to coronary artery disease and hypertension. On a population-based level, adult ECD is correlated to mortality from certain cardiovascular diseases. Clin. Anat. 31:927-936, 2018. © 2018 Wiley Periodicals, Inc.

Human axial progenitors generate trunk neural crest cells in vitro.

The neural crest (NC) is a multipotent embryonic cell population that generates distinct cell types in an axial position-dependent manner. The production of NC cells from human pluripotent stem cells (hPSCs) is a valuable approach to study human NC biology. However, the origin of human trunk NC remains undefined and current in vitro differentiation strategies induce only a modest yield of trunk NC cells. Here we show that hPSC-derived axial progenitors, the posteriorly-located drivers of embryonic axis elongation, give rise to trunk NC cells and their derivatives. Moreover, we define the molecular signatures associated with the emergence of human NC cells of distinct axial identities in vitro. Collectively, our findings indicate that there are two routes toward a human post-cranial NC state: the birth of cardiac and vagal NC is facilitated by retinoic acid-induced posteriorisation of an anterior precursor whereas trunk NC arises within a pool of posterior axial progenitors.

Abstract 575: Primary Cilia of Cardiac Neural Crest Cells Orchestrate Multiple Aspects of Cardiovascular Development

Cardiac neural crest cells (CNCC) are a specialized population of migratory neural crest cells that contribute to the developing cardiac outflow tract (OFT) and endocardial cushions (ECC). Like many eukaryotic cells, CNCC also possess primary cilia, which function as membrane-bound sensory organelles involved in intra- and extra-cellular communications in developing tissues. Previous work in our laboratory has shown that primary cilia of cardiac progenitor populations are critical orchestrators of both the septation and alignment of the developing OFT and interventricular septum. Our current in vivo models have refined the elimination of primary cilia to CNCC of the embryonic mouse heart using multiple neural crest-specific Cre drivers ( Wnt1 :Cre-Danielian et al,1998; Wnt1 :Cre2-Soriano et al, 2013) crossed with the Ift88 flox/flox mouse (Haycraft et al, 2007), resulting in elimination of primary cilia from CNCC during early embryonic heart development. Additionally, we have utilized a Td-Tomato (Tdt) reporter mouse line to track the migration of CNCC during various embryonic timepoints. Use of this reporter line revealed transient Tdt-positive CNCC contributions to the ventricular portion of the developing myocardium, in addition to the expected CNCC contributions to in the OFT and ECC regions. The resultant mutant phenotypes included a high incidence of outflow tract defects, impaired septation of the membranous interventricular septum, non-compaction of the developing ventricular myocardium, and perinatal lethality. Our data combine various immunohistochemical approaches to document, characterize, and quantify the novel presence of CNCC in the developing embryonic ventricular myocardium. These data suggest a potential modulatory role for primary cilia of CNCC in the development and maturation of the ventricular myocardium. Primary cilia-mediated signal transduction events that could account for the descried phenotypes are currently being elucidated.

Anatomy of the ventricular septal defect in congenital heart defects: a random association?

BackgroundA ventricular septal defect (VSD) is an integral part of most congenital heart defects (CHD). To determine the prevalence of VSD in various types of CHD and the distribution of their anatomic types.MethodsWe reviewed 1178 heart specimens with CHD from the anatomic collection of the French Reference Centre for Complex Congenital Heart Defects. During the morphologic study a special attention was paid to the localisation of the VSD viewed from the right ventricular side. The VSDs were classified as muscular, central perimembranous, outlet located between the two limbs of the septal band, and inlet. The specimens were classified according to the 9 categories and 23 subcategories of the anatomic and clinical classification of CHD1 (ACC-CHD).ResultsVentricular septum was almost always intact in anomalies of pulmonary veins (4/73, 5%), Ebstein anomaly (3/21, 14%), and double-inlet right ventricle (DIRV, 1/10, 10%). There was always a VSD in tetralogy of Fallot and variants (TOF, 123 cases) and common arterial trunk (CAT, 55 cases), always of the outlet type. There was almost always a VSD in double inlet left ventricle (33/34, 97%, always muscular), congenitally corrected transposition of great arteries (ccTGA, 23/24, 96%), interrupted aortic arch (IAA, 25/27, 93%), and double outlet right ventricle (DORV, 92/106, 87%). A VSD was found in 68% of aortic coarctation (CoA, 43/63), 62% of heterotaxy syndromes (21/34), 54% of transposition of the great arteries (TGA, 104/194). The VSD was located between the two limbs of the septal band in 100% of TOF and CAT, 80% of IAA, 77% of DORV, 82% of DD. The VSD was of the inlet type in 17% of cc TGA and in 71% of heterotaxy syndromes. In TGA, the VSD was outlet in 40%, central perimembranous in 25%, muscular in 25%, inlet in 10%. In CoA, the VSD was outlet in 44%, central perimembranous in 35%, muscular in 21%. In the 10% hearts with isolated VSD, the distribution was outlet in 44%, central perimembranous in 36%, muscular in 18%, and inlet in 2%.ConclusionThe anatomic distribution of VSD is similar in isolated VSD, CoA and TGA, while the VSD is predominantly outlet in outflow tract defects except TGA. This reinforces the allegedly different mechanisms in TGA and cardiac neural crest defects. This anatomic approach could provide new insights in the grouping and aetiology of CHD.