Neural Crest Cell Lineage Research Articles

Taste buds are not derived from neural crest in mouse, chicken, and zebrafish

Our lineage tracing studies using multiple Cre mouse lines showed a concurrent labeling of abundant taste bud cells and the underlying connective tissue with a neural crest (NC) origin, warranting a further examination on the issue of whether there is an NC derivation of taste bud cells. In this study, we mapped NC cell lineages in three different models, Sox10-iCreERT2/tdT mouse, GFP+ neural fold transplantation to GFP− chickens, and Sox10-Cre/GFP-RFP zebrafish model. We found that in mice, Sox10-iCreERT2 specifically labels NC cell lineages with a single dose of tamoxifen at E7.5 and that the labeled cells were widely distributed in the connective tissue of the tongue. No labeled cells were found in taste buds or the surrounding epithelium in the postnatal mice. In the GFP+/GFP− chicken chimera model, GFP+ cells migrated extensively to the cranial region of chicken embryos ipsilateral to the surgery side but were absent in taste buds in the base of oral cavity and palate. In zebrafish, Sox10-Cre/GFP-RFP faithfully labeled known NC-derived tissues but did not label taste buds in lower jaw or the barbel. Our data, together with previous findings in axolotl, indicate that taste buds are not derived from NC cells in rodents, birds, amphibians or teleost fish.

Quiescent Stem-Like Cells Drive NF1-Associated MPNST Functional Heterogeneity and Tumor Progression

NF1-associated malignant peripheral nerve sheath tumors (MPNST) are the major cause of mortality in neurofibromatosis. MPNST arise from benign peripheral nerve plexiform neurofibromas (PN) which originate in the embryonic neural crest cell lineage. Here we employ reporter transgenes that label early neural crest lineage cells in multiple NF1 MPNST mouse models to discover and characterize a rare MPNST cell population with stem cell-like properties including quiescence that is essential for tumor initiation and relapse. Following isolation of these cells we derive a cancer stem cell specific gene expression signature that includes consensus embryonic neural crest genes and also identify Nestin as a novel marker for the MPNST cell of origin. Combined targeting of cancer stem cells together with antimitotic chemotherapy yields effective tumor inhibition and prolongs survival. Finally, application of the mouse cancer stem cell signature to single cell sequencing data from a rare NF1-associated patient MPNST precursor tumor, identifies preformed unsupervised gene expression cell clusters. Enrichment of the cancer stem cell signature in cognate human tumors supports the generality and relevance of cancer stem cells to MPNST therapy development.

Pdgfra and Pdgfrb Genetically Interact in the Murine Neural Crest Cell Lineage to Regulate Migration and Proliferation.

Cranial neural crest cells (cNCCs) are migratory, multipotent cells that originate from the forebrain to the hindbrain and eventually give rise to the cartilage and bone of the frontonasal skeleton, among other derivatives. Signaling through the two members of the platelet-derived growth factor receptor (PDGFR) family of receptor tyrosine kinases, alpha and beta, plays critical roles in the cNCC lineage to regulate craniofacial development during murine embryogenesis. Further, the PDGFRs have been shown to genetically interact during murine craniofacial development at mid-to-late gestation. Here, we examined the effect of ablating both Pdgfra and Pdgfrb in the murine NCC lineage on earlier craniofacial development and determined the cellular mechanisms by which the observed phenotypes arose. Our results confirm a genetic interaction between the two receptors in this lineage, as phenotypes observed in an allelic series of mutant embryos often worsened with the addition of conditional alleles. The defects observed here appear to stem from aberrant cNCC migration, as well as decreased proliferation of the facial mesenchyme upon combined decreases in PDGFRα and PDGFRβ signaling. Importantly, we found that PDGFRα plays a predominant role in cNCC migration whereas PDGFRβ primarily contributes to proliferation of the facial mesenchyme past mid-gestation. Our findings provide insight into the distinct mechanisms by which PDGFRα and PDGFRβ signaling regulate cNCC activity and subsequent craniofacial development in the mouse embryo.

Investigate the Odontogenic Differentiation and Dentin-Pulp Tissue Regeneration Potential of Neural Crest Cells.

Stem cell-based developmental engineering has been considered as a promising strategy for tissue/organ regeneration. Tooth is formed by sequential reciprocal interactions between epithelium derived from surface ectoderm and mesenchymal cells derived from cranial neural crest. The neural crest cell is an appealing cell source for tooth development and regeneration research. In this study, we investigated the odontogenic differentiation and dentin-pulp complex regeneration potential of neural crest cells. Our results showed that neural crest cells (O9-1 mouse cranial neural crest cell line) can sequentially differentiate into dentin matrix acidic phosphoprotein 1 (DMP-1)-positive odontoblasts within a developing tooth germ in vitro. Moreover, O9-1 cells and induced pluripotent stem cell (iPSC)-derived neural crest-like cells (iNCLCs) can form well-organized vascularized dentin-pulp complex when transplanted in vivo with tooth scaffold. Furthermore, both O9-1 cells and iNCLCs can be differentiated into odontoblast-like cells, positive staining with odontogenic-related markers DMP-1 and dentin sialophosphoprotein (DSPP), under odontogenic induction with the administration of bone morphogenetic protein 4 (BMP-4). These results demonstrated that neural crest cells, especially the unlimited iNCLCs, are a promising cell source for tooth development and dental tissue/tooth organ regeneration studies.

Transforming Growth Factor-Beta and Sonic Hedgehog Signaling in Palatal Epithelium Regulate Tenascin-C Expression in Palatal Mesenchyme During Soft Palate Development.

During palatogenesis, the palatal shelves first grow vertically on either side of the tongue before changing their direction of growth to horizontal. The extracellular matrix (ECM) plays an important role in these dynamic changes in palatal shelf morphology. Tenascin-C (TNC) is an ECM glycoprotein that shows unique expression in the posterior part of the palatal shelf, but little is known about the regulation of TNC expression. Since transforming growth factor-beta-3 (TGF-β3) and sonic hedgehog (SHH) signaling are known to play important roles in palatogenesis, we investigated whether TGF-β3 and SHH are involved in the regulation of TNC expression in the developing palate. TGF-β3 increased the expression of TNC mRNA and protein in primary mouse embryonic palatal mesenchymal cells (MEPM) obtained from palatal mesenchyme dissected at embryonic day 13.5–14.0. Interestingly, immunohistochemistry experiments revealed that TNC expression was diminished in K14-cre;Tgfbr2fl/fl mice that lack the TGF-β type II receptor in palatal epithelial cells and exhibit cleft soft palate, whereas TNC expression was maintained in Wnt1-cre;Tgfbr2fl/fl mice that lack the TGF-β type II receptor in palatal mesenchymal cells and exhibit a complete cleft palate. SHH also increased the expression of TNC mRNA and protein in MEPM cells. However, although TGF-β3 up-regulated TNC mRNA and protein expression in O9-1 cells (a cranial neural crest cell line), SHH did not. Furthermore, TGF-β inhibited the expression of osteoblastic differentiation markers (osterix and alkaline phosphatase) and induced the expression of fibroblastic markers (fibronectin and periostin) in O9-1 cells, whereas SHH did not affect the expression of osteoblastic and fibroblastic markers in O9-1 cells. However, immunohistochemistry experiments showed that TNC expression was diminished in the posterior palatal shelves of Shh–/+;MFCS4+/– mice, which have deficient SHH signaling in the posterior palatal epithelium. Taken together, our findings support the proposal that TGF-β and SHH signaling in palatal epithelium co-ordinate the expression of TNC in the posterior palatal mesenchyme through a paracrine mechanism. This signal cascade may work in the later stage of palatogenesis when cranial neural crest cells have differentiated into fibroblast-like cells. The spatiotemporal regulation of ECM-related proteins by TGF-β and SHH signaling may contribute not only to tissue construction but also to cell differentiation or determination along the anterior–posterior axis of the palatal shelves.

KMT2D Haploinsufficiency in Kabuki Syndrome Impairs Differentiation of Neural Crest Cells

ObjectiveKabuki Syndrome (KS), caused by haploinsufficiency in lysine methyltransferase KMT2D, is a multi‐system disease characterized by distinct facial dysmorphisms, mild‐to‐moderate intellectual disability, renal and cardiac abnormalities, short stature and humoral immune deficiency. Many of the tissues in which these abnormalities manifest are derived from or developmentally modulated by the neural crest cell (NCC) lineage. This study’s objective was to determine which features of the KS phenotype can be directly linked to genetic and epigenetic dysregulation in KMT2D haploinsufficient NCCs.MethodsPeripheral blood mononuclear cells from a human donor haploinsufficient for KMT2D (KS patient, KMT2D+/−) or wild type (WT) control were used to generate induced pluripotent stem (iPS) cells. WT and KS NCCs were generated from WT and KS iPS cells, respectively. Whole transcriptome RNA sequencing (RNAseq) was performed on iPS cells (WT and KS) and NCCs (WT and KS) to characterize differential gene expression. Chromatin immunoprecipitation sequencing (ChIPseq) of histone 3 lysine 4 trimethylation (H3K4Me3) and histone 3 lysine 27 acetylation (H3K27Ac) was performed on NCCs (WT and KS) to characterize differential histone modifications at gene regulatory elements. Analysis of RNAseq and ChIPseq data was performed using BioWardrobe platform.ResultsGene set enrichment analysis of transcriptome data indicates that, compared to WT NCCs, KMT2D haploinsufficient NCCs have ectopic expression of several genes critical to normal NCC organization, morphogenesis and tissue differentiation, including NCC transcriptional repressor Msh homeobox 2 (MSX2). Analysis also identified several genes with decreased expression in KMT2D+/− NCCs, including transcription factor AP‐2 alpha (TFAP2A), which serves as a master regulator of NCC lineage progression. ChIPseq data indicate differential enrichment of H3K4Me3 and H3K27Ac histone modifications at the TFAP2A locus in WT compared to KS NCCs, which suggests that TFAP2A may have a direct role in the impaired differentiation of NCCs in KS. Further studies to explore the roles of TFAP2A and other genes identified by these data are underway.ConclusionThese results show that KMT2D haploinsufficiency impairs differentiation of the NCC lineage cells through epigenetic and transcriptomic dysregulation, thus supporting the classification of KS as a neurocristopathy.Support or Funding InformationNIH T32 Training Grant 5T32AI060515‐14 (WJL), R35 DE027557 (SB), Center for Pediatric Genomics Cincinnati Research Foundation (AWL).

Induction of Functional Mesenchymal Stem/Stromal Cells from Human iPCs Via a Neural Crest Cell Lineage Under Xeno-Free Conditions

Mesenchymal stem/stromal cells (MSCs) are adult multipotent stem cells. Here, we induced MSCs from human induced pluripotent stem cells (iPSCs) via a neural crest cell (NCC) lineage under xeno-free conditions and evaluated their in vivo functions. We modified a previous MSC induction method to work under xeno-free conditions. Bovine serum albumin-containing NCC induction medium and fetal bovine serum-containing MSC induction medium were replaced with xeno-free medium. Through our optimized method, iPSCs differentiated into MSCs with high efficiency. To evaluate their in vivo activities, we transplanted the xeno-free-induced MSCs (XF-iMSCs) into mouse models for bone and skeletal muscle regeneration and confirmed their regenerative potency. These XF-iMSCs mainly promoted the regeneration of surrounding host cells, suggesting that XF-iMSCs secrete soluble factors into affected regions. Notably, we found that the peroxidasin secreted by the XF-iMSCs contributed to myotube differentiation. These results suggest that XF-iMSCs are important for future applications in regenerative medicine.

Increased Fibronectin Impairs the Function of Excitatory/Inhibitory Synapses in Hirschsprung Disease.

Although approximately 50% of cases have a known genetic defect, the precise pathogenesis of Hirschsprung disease (HSCR) is still unclear. We recently reported that expression of fibronectin (FN), which is involved in the migration, colonization, and differentiation of enteric neural crest cells (ENCCs), is increased in aganglionic colonic segments obtained from patients. We hypothesized that abnormally high levels of FN might play a role in the etiology of HSCR. Here, to test this hypothesis, we investigated aganglionic, transitional, and ganglionic colon segments from 63 children with HSCR and distal colon from thirty healthy Wistar rats at embryonic day 20, in addition to in vitro studies with PC12 Adh neural crest cells. We measured the protein and mRNA expression levels of FN, together with a panel of excitatory (VGLUT1, GluA1, GluN1, PSD-95, and NL-1) and inhibitory (GAD67, GABA AR-α1, NL-2, and SLC32) synaptic markers. Expression of all these synaptic markers was significantly decreased in aganglionic colon, compared to ganglionic colon, whereas expression of FN was significantly increased. In a neural crest cell line, PC12 Adh, knockdown of FN with small-interfering RNA increased the expression of synaptic markers. Co-culture of colons from embryonic day 20 rats with RGD recombinant protein, which contains the RGD motif of FN, reduced the expression of excitatory and inhibitory synaptic markers. These results are consistent with the idea that the etiology of HSCR involves aberrant overexpression of FN, which may impair synaptic function and enteric nervous system development, leading to motor dysfunction of intestinal muscles.

MicroRNA-124-3p suppresses mouse lip mesenchymal cell proliferation through the regulation of genes associated with cleft lip in the mouse

BackgroundCleft lip (CL), one of the most common congenital birth defects, shows considerable geographic and ethnic variation, with contribution of both genetic and environmental factors. Mouse genetic studies have identified several CL-associated genes. However, it remains elusive how these CL-associated genes are regulated and involved in CL. Environmental factors may regulate these genes at the post-transcriptional level through the regulation of non-coding microRNAs (miRNAs). In this study, we sought to identify miRNAs associated with CL in mice.ResultsThrough a systematic literature review and a Mouse Genome Informatics (MGI) database search, we identified 55 genes that were associated with CL in mice. Subsequent bioinformatic analysis of these genes predicted that a total of 33 miRNAs target multiple CL-associated genes, with 20 CL-associated genes being potentially regulated by multiple miRNAs. To experimentally validate miRNA function in cell proliferation, we conducted cell proliferation/viability assays for the selected five candidate miRNAs (miR-124-3p, let-7a-5p, let-7b-5p, let-7c-5p, and let-7d-5p). Overexpression of miR-124-3p, but not of the others, inhibited cell proliferation through suppression of CL-associated genes in cultured mouse embryonic lip mesenchymal cells (MELM cells) isolated from the developing mouse lip region. By contrast, miR-124-3p knockdown had no effect on MELM cell proliferation. This miRNA-gene regulatory mechanism was mostly conserved in O9–1 cells, an established cranial neural crest cell line. Expression of miR-124-3p was low in the maxillary processes at E10.5, when lip mesenchymal cells proliferate, whereas it was greatly increased at later developmental stages, suggesting that miR-124-3p expression is suppressed during the proliferation phase in normal palate development.ConclusionsOur findings indicate that upregulated miR-124-3p inhibits cell proliferation in cultured lip cells through suppression of CL-associated genes. These results will have a significant impact, not only on our knowledge about lip morphogenesis, but also on the development of clinical approaches for the diagnosis and prevention of CL.

Hyaluronic acid is required for palatal shelf movement and its interaction with the tongue during palatal shelf elevation

Palatal shelf elevation is an essential morphogenetic process that results from palatal shelf movement caused by an intrinsic elevating force. The nature of the elevating force remains unclear, but the accumulation of hyaluronic acid (HA) in the extracellular matrix (ECM) of the palatal shelves may play a pivotal role in developing the elevating force. In mammals, HA is synthesized by hyaluronic acid synthases (HAS) that are encoded by three genes (Has1-3). Here, we used the Wnt1-Cre driver to conditionally disrupt hyaluronic acid synthase 2 (Has2) in cranial neural crest cell lineages. All Has2 conditional knockout (cko) mice had cleft palate due to failed shelf elevation during palate development. The HA content was significantly reduced in the craniofacial mesenchyme of Has2 cko mutants. Reduced HA content affected the ECM space and shelf expansion to result in a reduced shelf area and an increased mesenchymal cell density in the palatal shelves of Has2 cko mutants. We examined palatal shelf movement by removal of the tongue and mandible from unfixed E13.5 and early E14.5 embryonic heads. Reduced shelf expansion in Has2 cko mutants altered palatal shelf movement in the medial direction resulting in a larger gap between the palatal shelves than that of littermate controls. We further examined palatal shelf movement in the intact oral cavity by culturing explants containing the maxilla, palate, mandible and tongue (MPMT explants). The palatal shelves elevated alongside morphological changes in the tongue after 24-h culture in MPMT explants of early E14.5 wild type embryos. On the contrary, shelf elevation failed to occur in MPMT explants of age-matched Has2 cko mutants because the tongue obstructs palatal shelf movement, suggesting that reduced shelf expansion could be essential for the palatal shelves to interact with the tongue and overcome tongue obstruction during shelf elevation. Has2 cko mutants also showed micrognathia due to reduced HA content in the mandibular mesenchyme including Meckel’s cartilage. Through 3D imaging and morphometric analysis, we demonstrate that mandibular growth results in a significant increase in the vertical dimension of the common oral-nasal cavity that facilitates palatal shelf movement and its interaction with the tongue during shelf elevation.

Ex Vivo Culture and Manipulation of Mouse Neural Crest Cells from Primary Embryonic Tissue Explants.

In vitro culture of neural crest cells allows for the manipulation and study of neural crest cell function in a cell-autonomous manner. While several stable neural crest cell lines exist, the transformed nature of these cells may not closely reflect the in vivo properties of neural crest cells, hence making molecular and functional analyses using these cell lines difficult to interpret. Here we describe a robust method to culture primary mouse neural crest cells ex vivo for several days to weeks in culture. We further describe a method for siRNA knockdown in these cells to study gene function. This culture method can also be adapted for other molecular analyses, including addition of small-molecule inhibitors and/or growth factors to the culture media, as well as culturing neural crest cells from knockout or genetically modified mice.

Nerve growth factor prevents arsenic-induced toxicity in PC12 cells through the AKT/GSK-3β/NFAT pathway.

The potential risk of arsenic-related neurodegeneration has been a growing concern. Arsenic exposure has been reported to disrupt neurite growth and neuron body integrity in vitro; however, its underlying mechanism remains unclear. Previously, we showed that arsenic sulfide (AS) exerted cytotoxicity in gastric and colon cancer cells through regulating nuclear factor of the activated T cells (NFAT) pathway. The NFAT pathway regulates axon path finding and neural development. Using neural crest cell line PC12 cells as a model, here we show that AS caused mitochondrial membrane potential collapse, reactive oxygen species production, and cytochrome c release, leading to mitochondria-mediated apoptosis via the AKT/GSK-3β/NFAT pathway. Increased glycogen synthase kinase-3 beta (GSK-3β) activation leads to the inactivation of NFAT and its antiapoptotic effects. Through inhibiting GSK-3β activity, both nerve growth factor (NGF) and Tideglusib, a GSK-3β inhibitor partially rescued the PC12 cells from the AS-induced cytotoxicity and restored the expression of NFATc3. In addition, overexpression of NFATc3 stimulated neurite outgrowth and potentiated the effect of NGF on promoting the neurite outgrowth. Collectively, our results show that NFATc3 serves as the downstream target of NGF and plays a key role in preventing AS-induced neurotoxicity through regulating the AKT/GSK-3β/NFAT pathway in PC12 cells.

TFII-I and AP2α Co-Occupy the Promoters of Key Regulatory Genes Associated with Craniofacial Development.

The aim of this study is to define the candidate target genes for TFII-I and AP2α regulation in neural crest progenitor cells. The GTF2I and GTF2IRD1 genes encoding the TFII-I family of transcription factors are prime candidates for the Williams-Beuren syndrome, a complex multisystem disorder characterized by craniofacial, skeletal, and neurocognitive deficiencies. AP2α, a product of the TFAP2A gene, is a master regulator of neural crest cell lineage. Mutations in TFAP2A cause branchio-oculo-facial syndrome characterized by dysmorphic facial features and orofacial clefts. In this study, we examined the genome-wide promoter occupancy of TFII-I and AP2α in neural crest progenitor cells derived from in vitro-differentiated human embryonic stem cells. Our study revealed that TFII-I and AP2α co-occupy a selective set of genes that control the specification of neural crest cells. The data suggest that TFII-I and AP2α may coordinately control the expression of genes encoding chromatin-modifying proteins, epigenetic enzymes, transcription factors, and signaling proteins.

Transcription factor Foxc1 is involved in anterior part of cranial base formation.

The cranial base is a structure mainly formed through endochondral ossification and integrated into the craniofacial complex, which acts as an underlying platform for the developing brain. Foxc1 is an indispensable regulator during intramembranous and endochondral ossification. In this study, we found that the spontaneous loss of Foxc1 function in a mouse (congenital hydrocephalous), Foxc1ch/ch , demonstrated the anterior cranial base defects, including unossified presphenoid and lack of middle part of the basisphenoid bone. Hypoplastic presphenoid primordial cartilage (basal portion of the trabecular cartilage [bTB]) and a lack of the middle part of basisphenoid primordial cartilage (the hypophyseal cartilage) were consistently observed at earlier developmental stage. Foxc1 was expressed robustly and ubiquitously in undifferentiated mesenchyme of the cranial base-forming area in E11.0 wild-type fetuses. Once chondrogenesis commenced, the expression was downregulated and later limited to the perichondrium. Detection of transcripts of Collagen type2 A1 (Col2a1) revealed that both bTB and the anterior part of the hypophyseal cartilage developing anterior to the persistent epithelial stalk of the anterior lobe of the pituitary gland were suppressed in the Foxc1ch/ch . Proliferation activity of chondrocyte precursor cells was higher in the Foxc1ch/ch . Loss of Foxc1 function only in the neural crest cell lineage (Wnt1-cre;Foxc1ch/flox ) showed ossification of the posterior part of the hypophyseal cartilage derived from the mesoderm. These findings suggest that Foxc1 is an important regulator to further chondrogenesis and initiate the ossification of the presphenoid and basisphenoid bones.

E-cigarette aerosol exposure can cause craniofacial defects in Xenopus laevis embryos and mammalian neural crest cells.

Since electronic cigarette (ECIG) introduction to American markets in 2007, vaping has surged in popularity. Many, including women of reproductive age, also believe that ECIG use is safer than traditional tobacco cigarettes and is not hazardous when pregnant. However, there are few studies investigating the effects of ECIG exposure on the developing embryo and nothing is known about potential effects on craniofacial development. Therefore, we have tested the effects of several aerosolized e-cigarette liquids (e-cigAM) in an in vivo craniofacial model, Xenopus laevis, as well as a mammalian neural crest cell line. Results demonstrate that e-cigAM exposure during embryonic development induces a variety of defects, including median facial clefts and midface hypoplasia in two of e-cigAMs tested e-cigAMs. Detailed quantitative analyses of the facial morphology revealed that nicotine is not the main factor in inducing craniofacial defects, but can exacerbate the effects of the other e-liquid components. Additionally, while two different e-cigAMs can have very similar consequences on facial appearances, there are subtle differences that could be due to the differences in e-cigAM components. Further assessment of embryos exposed to these particular e-cigAMs revealed cranial cartilage and muscle defects and a reduction in the blood supply to the face. Finally, the expression of markers for vascular and cartilage differentiation was reduced in a mammalian neural crest cell line corroborating the in vivo effects. Our work is the first to show that ECIG use could pose a potential hazard to the developing embryo and cause craniofacial birth defects. This emphasizes the need for more testing and regulation of this new popular product.

Abstract LB-083: Core transcriptional regulatory circuitries in neuroblastoma

Abstract Background Neuroblastoma (NB) is a pediatric tumor derived from precursor cells of the sympathetic nervous system. NB accounts for 12% of all childhood cancer deaths with ~50% high-risk cases which frequently harbor amplified proto-oncogene MYCN. Evidence accumulates that epigenetic deregulation, including aberrant DNA methylation in high-risk disease or oncogene activation by enhancer hijacking, plays a prominent role in NB. The present study applies a comprehensive approach integrating chromatin modification data with genomic and expression data to elucidate NB subtype specific super-enhancer (SE) landscapes and core regulatory circuitries (CRCs) consisting of lineage-specific interconnected loops of SE-driven, auto-regulatory master transcription factors. Methods Chromatin immunoprecipitation sequencing (ChIP-seq) of histone 3 lysine 27 acetylation (H3K27ac) was used to identify active enhancer elements in 23 primary NBs. A validation cohort consisting of 16 NB cell lines and two human neural crest cell lines was used. ChIPmentation was applied to validate predicted transcription factor (TF) binding events. Circular chromatin conformation capture sequencing (4C-seq) was used to assay physical promoter-enhancer interactions. Results Unsupervised clustering of 23 primary NBs according to H3K27ac signal intensity at the most variable SEs (genome-wide) revealed two main subgroups, MYCN-amplified (n = 8) and MYCN single copy tumors (n = 15), with distinctive activity patterns. Calling of CRCs in the 23 primary NBs yielded a core set of NB master TFs (CRC TFs). Amongst the top ten of them are HAND2, PHOX2B and MYCN, all of which are implicated in NB biology and playing essential roles in the development of the sympathetic nervous system. In line with this, gene ontology analyses of the top 50 CRC TFs converge on biological processes like development of neural crest cells, sympathetic nervous system and peripheral nervous system neurons. ChIPmentation analyses of selected CRC TFs confirmed auto-binding to their assigned SEs and those of other CRC TFs in their respective network. Interactions between promoters and SEs of selected CRC TFs were verified via 4C-seq. Intriguingly, expression analysis of the top 50 CRC TFs in a cohort of 498 primary NBs revealed that less than 20% of the CRC TFs are up-regulated in MYCN-amplified tumors while the remaining 80% are down-regulated in that subgroup. This suggests a superordinate role of MYCN in differentially orchestrating NB master TFs. Conclusion The study identifies the core set of NB master transcription factors and assigns established NB regulators like HAND2, PHOX2B and MYCN to well-defined CRCs. It reveals an association of MYCN amplification with the global SE landscape of primary NBs and suggests a role for MYCN in differentially controlling subsets of CRC TFs and their networks. Specific targeting of the SE-dependent CRC networks may open a therapeutic window for epigenetic drugs, including BET inhibitors, CDK7 or EZH2 inhibition, in NB. Citation Format: Moritz Gartlgruber, Daniel Dreidax, Daria Doncevic, Sebastian Steinhauser, Stefan Gröschel, Kai Oliver Henrich, Young-Gyu Park, Carl Herrmann, Frank Westermann. Core transcriptional regulatory circuitries in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-083. doi:10.1158/1538-7445.AM2017-LB-083

Recent biologic and genetic advances in neuroblastoma: Implications for diagnostic, risk stratification, and treatment strategies

Neuroblastoma is an embryonic cancer of neural crest cell lineage, accounting for up to 10% of all pediatric cancer. The clinical course is heterogeneous ranging from spontaneous regression in neonates to life-threatening metastatic disease in older children. Much of this clinical variance is thought to result from distinct pathologic characteristics that predict patient outcomes. Consequently, many research efforts have been focused on identifying the underlying biologic and genetic features of neuroblastoma tumors in order to more clearly define prognostic subgroups for treatment stratification. Recent technological advances have placed emphasis on the integration of genetic alterations and predictive biologic variables into targeted treatment approaches to improve patient survival outcomes. This review will focus on these recent advances and the implications they have on the diagnostic, staging, and treatment approaches in modern neuroblastoma clinical management.

Phenotypes associated with psychiatric disorders are sex-specific in a mutant mouse line

Wnt1-Cre- and Wnt1-GAL4 double transgenic (dTg) mice are used to study neural crest cell lineages by utilizing either the Cre/loxP or the GAL4/UAS system. We have previously shown that these mice exhibit behavioral abnormalities that resemble certain behaviors of psychiatric disorders and histologic alterations in the cholinergic and glutamatergic systems in the brain. The objective of the current study was to extend the behavioral analyses in these mice and to determine whether there were any sex-specific differences in the prevalence or severity of these behaviors. In the present study, we demonstrate additional behavioral abnormalities in dTg mice, such as increased locomotor activity, decreased social behavior, and an increased frequency in vertical jumping. Of these, the proclivity for vertical jumping was observed only in male dTg mice. In contrast, MK-801 administration induced increased locomotion in only female dTg mice. Furthermore, the concentrations of prolactin in the sera and oxytocin in the hypothalamus were both reduced only in female dTg mice, compared to controls. These sex-dependent behavioral and hormonal abnormalities in the dTG mice suggest that the phenotype of certain psychiatric disorders may be influenced by both genetic and sex-specific factors.

Abstract 323: Loss of Gravity Impairs Cardiac Neural Crest Cell Lineage Development and Function

Introduction: A multitude of structural, haemodynamic and electromechanical cardiovascular disorders have been observed in humans following space-travel. These abnormalities are thought to emerge from transient alterations in autonomic nervous system (ANS). However, since the ANS is cardiac neural crest (CNC)-derived, whether microgravity-induced cardiomyopathies reflect CNC dysfunction, is unknown. Hypothesis: Impairment of CNCs underlies microgravity-induced cardiomyopathies. Methods: Myocardial explants from adult cKit CreERT2/+ ;IRG mice (n=5/group), as well as cKit CreERT2/+ ;IRG- derived (iPSC Kit-Cre ; n=6/group) and Wnt1-Cre;tdTomato -derived (iPSC Wnt1-Cre ; n=18/group) induced pluripotent stem cells, were cultured under static (SC) or simulated microgravity conditions (rotary cell-culture system; RCCS). Results: CNC lineage-tracing in cardiac explants illustrated that, compared to SC, RCCS abolished the pool of cKit + CNCs in adult hearts, indicated by quantitation of cKit CreERT2 - mediated EGFP expression ( p <0.05). Cardiogenesis modeling experiments with iPSC Kit-Cre yielded fewer beating EBs ( p =0.0005), and ~10-fold reduction in EGFP + cardiomyocytes ( p =0.01), in RCCS vs . SC. Microarray analyses suggested that RCCS-mediated alterations in BMP and Wnt/β-catenin pathways, downregulated ANS and CNC-related gene programs, and enhanced vasculogenic differentiation without affecting the expression of cardiac mesoderm-related genes. Differences were verified by quantitative PCR. Modeling CNC development in iPSC Wnt1-Cre further confirmed an RCCS-mediated dramatic impairment in development and function of CNCs, indicated by quantitation of tdTomato expression in day-10 and day-21 beating embryoid bodies ( p <0.0001). Intriguingly, the effect of RCCS in CNCs could be only partially rescued upon transfer to SC. Conclusions: Together these data indicate that microgravity negatively regulates the development and function of CNCs, thus partly explaining the cellular and molecular mechanisms of microgravity-induced cardiomyopathies. Moreover, these findings are expected to have important implications in space exploration, since they suggest an essential role for gravity in vertebrate development.

SS18-SSX, the Oncogenic Fusion Protein in Synovial Sarcoma, Is a Cellular Context-Dependent Epigenetic Modifier.

The prevalence and specificity of unique fusion oncogenes are high in a number of soft tissue sarcomas (STSs). The close relationship between fusion genes and clinicopathological features suggests that a correlation may exist between the function of fusion proteins and cellular context of the cell-of-origin of each tumor. However, most STSs are origin-unknown tumors and this issue has not yet been investigated in detail. In the present study, we examined the effects of the cellular context on the function of the synovial sarcoma (SS)-specific fusion protein, SS18-SSX, using human pluripotent stem cells (hPSCs) containing the drug-inducible SS18-SSX gene. We selected the neural crest cell (NCC) lineage for the first trial of this system, induced SS18-SSX at various differentiation stages from PSCs to NCC-derived mesenchymal stromal cells (MSCs), and compared its biological effects on each cell type. We found that the expression of FZD10, identified as an SS-specific gene, was induced by SS18-SSX at the PSC and NCC stages, but not at the MSC stage. This stage-specific induction of FZD10 correlated with stage-specific changes in histone marks associated with the FZD10 locus and also with the loss of the BAF47 protein, a member of the SWI/SNF chromatin-remodeling complex. Furthermore, the global gene expression profile of hPSC-derived NCCs was the closest to that of SS cell lines after the induction of SS18-SSX. These results clearly demonstrated that the cellular context is an important factor in the function of SS18-SSX as an epigenetic modifier.