Cardiac Neural Crest Cell Migration Research Articles

Gene-Interaction-Sensitive enrichment analysis in congenital heart disease

BackgroundGene set enrichment analysis (GSEA) uses gene-level univariate associations to identify gene set-phenotype associations for hypothesis generation and interpretation. We propose that GSEA can be adapted to incorporate SNP and gene-level interactions. To this end, gene scores are derived by Relief-based feature importance algorithms that efficiently detect both univariate and interaction effects (MultiSURF) or exclusively interaction effects (MultiSURF*). We compare these interaction-sensitive GSEA approaches to traditional χ2 rankings in simulated genome-wide array data, and in a target and replication cohort of congenital heart disease patients with conotruncal defects (CTDs).ResultsIn the simulation study and for both CTD datasets, both Relief-based approaches to GSEA captured more relevant and significant gene ontology terms compared to the univariate GSEA. Key terms and themes of interest include cell adhesion, migration, and signaling. A leading edge analysis highlighted semaphorins and their receptors, the Slit-Robo pathway, and other genes with roles in the secondary heart field and outflow tract development.ConclusionsOur results indicate that interaction-sensitive approaches to enrichment analysis can improve upon traditional univariate GSEA. This approach replicated univariate findings and identified additional and more robust support for the role of the secondary heart field and cardiac neural crest cell migration in the development of CTDs.

Prdm6 controls heart development by regulating neural crest cell differentiation and migration.

The molecular mechanisms that drive the acquisition of distinct neural crest cell (NCC) fates is still poorly understood. Here, we identified Prdm6 as an epigenetic modifier that temporally and spatially regulates the expression of NCC specifiers and determines the fate of a subset of migrating cardiac NCCs (CNCCs). Using transcriptomic analysis and genetic and fate mapping approaches in transgenic mice, we showed that disruption of Prdm6 was associated with impaired CNCC differentiation, delamination, and migration and led to patent ductus arteriosus (DA) and ventricular noncompaction. Bulk and single-cell RNA-Seq analyses of the DA and CNCCs identified Prdm6 as a regulator of a network of CNCC specification genes, including Wnt1, Tfap2b, and Sox9. Loss of Prdm6 in CNCCs diminished its expression in the pre-epithelial–mesenchymal transition (pre-EMT) cluster, resulting in the retention of NCCs in the dorsal neural tube. This defect was associated with diminished H4K20 monomethylation and G1-S progression and augmented Wnt1 transcript levels in pre-EMT and neural tube clusters, which we showed was the major driver of the impaired CNCC migration. Altogether, these findings revealed Prdm6 as a key regulator of CNCC differentiation and migration and identified Prdm6 and its regulated network as potential targets for the treatment of congenital heart diseases.

Coadministration of ARV (Atripla) and Topiramate disrupts quail cardiac neural crest cell migration.

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. 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. 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. These results indicate that Atripla and Topiramate cause ventricular septal defects by inhibiting the migration of cardiac neural crest cells.

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.

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 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.

Role of Semaphorin Signaling During Cardiovascular Development.

Semaphorins are a family of secreted and membrane‐bound molecules that play critical functions in diverse biological processes.[1][1], [2][2], [3][3], [4][4], [5][5], [6][6], [7][7], [8][8], [9][9] SEMA1A/Fasciclin IV is the founding member of the semaphorin family and was first reported in 1992

Endogenous Natural Complement Inhibitor Regulates Cardiac Development.

Congenital heart defects are a major cause of perinatal mortality and morbidity, affecting >1% of all live births in the Western world, yet a large fraction of such defects have an unknown etiology. Recent studies demonstrated surprising dual roles for immune-related molecules and their effector mechanisms during fetal development and adult homeostasis. In this article, we describe the function of an endogenous complement inhibitor, mannan-binding lectin (MBL)-associated protein (MAp)44, in regulating the composition of a serine protease-pattern recognition receptor complex, MBL-associated serine protease (MASP)-3/collectin-L1/K1 hetero-oligomer, which impacts cardiac neural crest cell migration. We used knockdown and rescue strategies in zebrafish, a model allowing visualization and assessment of heart function, even in the presence of severe functional defects. Knockdown of embryonic expression of MAp44 caused impaired cardiogenesis, lowered heart rate, and decreased cardiac output. These defects were associated with aberrant neural crest cell behavior. We found that MAp44 competed with MASP-3 for pattern recognition molecule interaction, and knockdown of endogenous MAp44 expression could be rescued by overexpression of wild-type MAp44. Our observations provide evidence that immune molecules are centrally involved in the orchestration of cardiac tissue development.

Zebrafish heart development is regulated via glutaredoxin 2 dependent migration and survival of neural crest cells

Glutaredoxin 2 is a vertebrate specific oxidoreductase of the thioredoxin family of proteins modulating the intracellular thiol pool. Thereby, glutaredoxin 2 is important for specific redox signaling and regulates embryonic development of brain and vasculature via reversible oxidative posttranslational thiol modifications. Here, we describe that glutaredoxin 2 is also required for successful heart formation. Knock-down of glutaredoxin 2 in zebrafish embryos inhibits the invasion of cardiac neural crest cells into the primary heart field. This leads to impaired heart looping and subsequent obstructed blood flow. Glutaredoxin 2 specificity of the observed phenotype was confirmed by rescue experiments. Active site variants of glutaredoxin 2 revealed that the (de)-glutathionylation activity is required for proper heart formation. Our data suggest that actin might be one target during glutaredoxin 2 regulated cardiac neural crest cell migration and embryonic heart development. In summary, this work represents further evidence for the general importance of redox signaling in embryonic development and highlights additionally the importance of glutaredoxin 2 during embryogenesis.

Misregulation of SDF1-CXCR4 Signaling Impairs Early Cardiac Neural Crest Cell Migration Leading to Conotruncal Defects

Cardiac neural crest cells (NCs) contribute to heart morphogenesis by giving rise to a variety of cell types from mesenchyme of the outflow tract, ventricular septum, and semilunar valves to neurons of the cardiac ganglia and smooth muscles of the great arteries. Failure in cardiac NC development results in outflow and ventricular septation defects commonly observed in congenital heart diseases. Cardiac NCs derive from the vagal neural tube, which also gives rise to enteric NCs that colonize the gut; however, so far, molecular mechanisms segregating these 2 populations and driving cardiac NC migration toward the heart have remained elusive. Stromal-derived factor-1 (SDF1) is a chemokine that mediates oriented migration of multiple embryonic cells and mice deficient for Sdf1 or its receptors, Cxcr4 and Cxcr7, exhibit ventricular septum defects, raising the possibility that SDF1 might selectively drive cardiac NC migration toward the heart via a chemotactic mechanism. We show in the chick embryo that Sdf1 expression is tightly coordinated with the progression of cardiac NCs expressing Cxcr4. Cxcr4 loss-of-function causes delayed migration and enhanced death of cardiac NCs, whereas Sdf1 misexpression results in their diversion from their normal pathway, indicating that SDF1 acts as a chemoattractant for cardiac NCs. These alterations of SDF1 signaling result in severe cardiovascular defects. These data identify Sdf1 and its receptor Cxcr4 as candidate genes responsible for cardiac congenital pathologies in human.

Cardiac neural crest is dispensable for outflow tract septation in Xenopus

In vertebrate embryos, cardiac precursor cells of the primary heart field are specified in the lateral mesoderm. These cells converge at the ventral midline to form the linear heart tube, and give rise to the atria and the left ventricle. The right ventricle and the outflow tract are derived from an adjacent population of precursors known as the second heart field. In addition, the cardiac neural crest contributes cells to the septum of the outflow tract to separate the systemic and the pulmonary circulations. The amphibian heart has a single ventricle and an outflow tract with an incomplete spiral septum; however, it is unknown whether the cardiac neural crest is also involved in outflow tract septation, as in amniotes. Using a combination of tissue transplantations and molecular analyses in Xenopus we show that the amphibian outflow tract is derived from a second heart field equivalent to that described in birds and mammals. However, in contrast to what we see in amniotes, it is the second heart field and not the cardiac neural crest that forms the septum of the amphibian outflow tract. In Xenopus, cardiac neural crest cells remain confined to the aortic sac and arch arteries and never populate the outflow tract cushions. This significant difference suggests that cardiac neural crest cell migration into the cardiac cushions is an amniote-specific characteristic, presumably acquired to increase the mass of the outflow tract septum with the evolutionary need for a fully divided circulation.

Cardiac neural crest is dispensable for outflow tract septation in Xenopus

In vertebrate embryos, cardiac precursor cells of the primary heart field give rise to the atria and the left ventricle. The right ventricle and the outflow tract are later evolutionary innovations derived from an adjacent population of precursors known as the second heart field. In addition, the cardiac neural crest contributes cells to the septum of the outflow tract to separate the systemic and the pulmonary circulations. The amphibian heart lacks a right ventricle but has a rudimentary outflow tract and a spiral septum, however it is unknown whether the cardiac neural crest is also involved in outflow tract septation as seen in amniotes. Using a combination of tissue transplantations and molecular analyses in Xenopus we show that the amphibian outflow tract is derived from a second heart field equivalent to the one described in birds and mammals. However, in contrast to what we see in amniotes it is the second heart field and not the cardiac neural crest that forms the septum of the amphibian outflow tract. In Xenopus cardiac neural crest cells remain confined to the aortic sac and arch arteries and never populate the outflow tract cushions. This significant difference suggests that cardiac neural crest cell migration into the cardiac cushions is an amniote-specific characteristic, presumably acquired to increase the mass of the outflow tract septum with the evolutionary need for a fully divided circulation.

Dicer activity in neural crest cells is essential for craniofacial organogenesis and pharyngeal arch artery morphogenesis

MicroRNAs (miRNAs) play important roles in regulating gene expression during numerous biological/pathological processes. Dicer encodes an RNase III endonuclease that is essential for generating most, if not all, functional miRNAs. In this work, we applied a conditional gene inactivation approach to examine the function of Dicer during neural crest cell (NCC) development. Mice with NCC-specific inactivation of Dicer died perinatally. Cranial and cardiac NCC migration into target tissues was not affected by Dicer disruption, but their subsequent development was disturbed. NCC derivatives and their associated mesoderm-derived cells displayed massive apoptosis, leading to severe abnormalities during craniofacial morphogenesis and organogenesis. In addition, the 4th pharyngeal arch artery (PAA) remodeling was affected, resulting in interrupted aortic arch artery type B (IAA-B) in mutant animals. Taken together, our results show that Dicer activity in NCCs is essential for craniofacial development and pharyngeal arch artery morphogenesis.

Absence of the vagus nerve in the stomach of Tbx1−/− mutant mice

Tbx1 is a member of the Tbox family of binding domain transcription factors. TBX1 maps within the region of chromosome 22q11 deleted in humans with DiGeorge syndrome (DGS), a common genetic disorder characterized by numerous physical manifestations including craniofacial and cardiac anomalies. Mice with homozygous null mutations in Tbx1 phenocopy this disorder and have defects including abnormal cranial ganglia formation and cardiac neural crest cell migration. These defects prompted us to investigate whether extrinsic vagus nerve or intrinsic enteric nervous system abnormalities are prevalent in the gastrointestinal tract of Tbx1 mutant mice. We used in situ hybridization for Ret, and immunohistochemical staining for neurofilament, HuC/D and βIII-tubulin to study cranial ganglia, vagus nerve, and enteric nervous system development in Tbx1 mutant and control mice. In Tbx1(-/-) embryos, cranial ganglia of the glossopharyngeal (IXth) and vagus (Xth) nerves were malformed and abnormally fused. In the gastrointestinal tract, the vagus nerves adjacent to the esophagus were severely hypoplastic and they did not extend beyond the gastro-esophageal junction nor project branches within the stomach wall, as was observed in Tbx1(+/+) mice. Although cranial ganglia morphology appeared normal in Tbx1(+/-) mice, these animals had a spectrum of stomach vagus innervation defects ranging from mild to severe. In all Tbx1 genotypes, the intrinsic enteric nervous system developed normally. The deficit in vagal innervation of the stomach in mice mutant for a gene implicated in DGS raises the possibility that similar defects may underlie a number of as yet unidentified/unreported congenital disorders affecting gastrointestinal function.

Tbx1 controls cardiac neural crest cell migration during arch artery development by regulatingGbx2expression in the pharyngeal ectoderm

Elucidating the gene regulatory networks that govern pharyngeal arch artery (PAA) development is an important goal, as such knowledge can help to identify new genes involved in cardiovascular disease. The transcription factor Tbx1 plays a vital role in PAA development and is a major contributor to cardiovascular disease associated with DiGeorge syndrome. In this report, we used various genetic approaches to reveal part of a signalling network by which Tbx1 controls PAA development in mice. We investigated the crucial role played by the homeobox-containing transcription factor Gbx2 downstream of Tbx1. We found that PAA formation requires the pharyngeal surface ectoderm as a key signalling centre from which Gbx2, in response to Tbx1, triggers essential directional cues to the adjacent cardiac neural crest cells (cNCCs) en route to the caudal PAAs. Abrogation of this signal generates cNCC patterning defects leading to PAA abnormalities. Finally, we showed that the Slit/Robo signalling pathway is activated during cNCC migration and that components of this pathway are affected in Gbx2 and Tbx1 mutant embryos at the time of PAA development. We propose that the spatiotemporal control of this tightly orchestrated network of genes participates in crucial aspects of PAA development.

13-P005 Tbx1 controls cardiac neural crest cell migration during arch artery development by regulating Gbx2 expression in the pharyngeal ectoderm

13-P005 Tbx1 controls cardiac neural crest cell migration during arch artery development by regulating Gbx2 expression in the pharyngeal ectoderm

A MMP‐independent role for TIMP‐2 during cardiac neural crest cell migration

Tissue inhibitor of metalloproteinase‐2 (TIMP‐2), a matrix metalloproteinase (MMP) inhibitor, is uniquely expressed in cardiac neural crest (NC) cells. Blocking TIMP‐2 synthesis perturbs NC migration while synthetic MMP inhibitors have little effect suggesting TIMP‐2 operates independent of MMP. TIMP‐2 can mediate growth factor‐induced responses independent of MMP inhibition by binding α3ß1 integrins. Here, we compared the effects of wild‐type TIMP‐2, a non‐MMP inhibiting form of TIMP‐2 (Ala+TIMP‐2), and a TIMP‐2/integrin blocking peptide on NC migratory behavior. Ala+TIMP‐2 increased NC cell motility, increased the distance NC cells migrated from neural tube explants, and altered NC cell shape (the cells were flatter and more arborized) compared to control cultures. Wild‐type TIMP‐2 also increased NC motility but had no effect on the distance NC cells migrated nor on their cell shape. A TIMP‐2/integrin inhibitory peptide had no effect on basal NC migration but it blocked the effects of Ala+TIMP‐2 in vitro and perturbed NC migration when injected into embryos. In addition, α3 integrin protein was expressed by the ectoderm, neural tube, and NC cells in situ. These data suggest TIMP‐2's functional role in NC migration may be mediated through α3ß1 integrin signaling rather than inhibiting MMP activity. Supported by the American Heart Association, Heartland Affiliate (#0555683Z) and NIH (#P20‐RR016469).

Cooperation between the PDGF receptors in cardiac neural crest cell migration

Neural crest cells (NCCs) are essential components of the sympathetic nervous system, skin, craniofacial skeleton, and aortic arch. It has been known for many years that perturbation of migration, proliferation, and/or differentiation of these cells leads to birth defects such as cleft palate and persistent truncus arteriosus (PTA). Previously, we had shown that disruption of the platelet-derived growth factor receptor (PDGFR) α in NCCs resulted in defects in craniofacial and aortic arch development, the latter with variable penetrance. Because we observed ventricular septal defects in embryos that are null for the PDGFRβ, we hypothesized that both PDGF receptors are involved in NCC formation. Here, we show that both receptors are expressed in cardiac NCCs and that the combined loss of the PDGFRα and PDGFRβ in NCCs resulted in NCC-related heart abnormalities, including PTA and a ventricular septal defect (VSD). Using NCC lineage tracing, we observed that loss of PDGF receptor signaling resulted in reduced NCCs in the conotruncus region, leading to defects in aortic arch septation. These results indicate that while PDGFRα plays a predominant role in NCC development, the PDGFRβ is expressed by and functions in cardiac NCCs. Combined PDGF receptor signaling is required for sufficient recruitment of cardiac NCCs into the conotruncal region and for formation of the aortico-pulmonary and ventricular septum.

Connexin 43-mediated modulation of polarized cell movement and the directional migration of cardiac neural crest cells

Connexin 43 knockout (Cx43alpha1KO) mice have conotruncal heart defects that are associated with a reduction in the abundance of cardiac neural crest cells (CNCs) targeted to the heart. In this study, we show CNCs can respond to changing fibronectin matrix density by adjusting their migratory behavior, with directionality increasing and speed decreasing with increasing fibronectin density. However, compared with wild-type CNCs, Cx43alpha1KO CNCs show reduced directionality and speed, while CNCs overexpressing Cx43alpha1 from the CMV43 transgenic mice show increased directionality and speed. Altered integrin signaling was indicated by changes in the distribution of vinculin containing focal contacts, and altered temporal response of Cx43alpha1KO and CMV43 CNCs to beta1 integrin function blocking antibody treatment. High resolution motion analysis showed Cx43alpha1KO CNCs have increased cell protrusive activity accompanied by the loss of polarized cell movement. They exhibited an unusual polygonal arrangement of actin stress fibers that indicated a profound change in cytoskeletal organization. Semaphorin 3A, a chemorepellent known to inhibit integrin activation, was found to inhibit CNC motility, but in the Cx43alpha1KO and CMV43 CNCs, cell processes failed to retract with semaphorin 3A treatment. Immunohistochemical and biochemical analyses suggested close interactions between Cx43alpha1, vinculin and other actin-binding proteins. However, dye coupling analysis showed no correlation between gap junction communication level and fibronectin plating density. Overall, these findings indicate Cx43alpha1 may have a novel function in mediating crosstalk with cell signaling pathways that regulate polarized cell movement essential for the directional migration of CNCs.