Neural Crest Markers Research Articles

Identification and characterization of chromaffin progenitor cells in murine adrenal medulla

Background: The adrenal gland exhibits a high plasticity and chromaffin cell number can be regulated, in contrast to the closely related neurons, to meet physiological needs. It therefore has been suggested that a subpopulation of proliferation and differentiation competent progenitor cells continues to exist in the adult adrenal medulla. In our previous studies, we established protocols to in vitro enrich these cells from bovine and human adrenals in sphere cultures. These cells are characterized by the expression of neural crest and neural progenitor markers and are able to differentiate to the chromaffin and neural lineage.

Beta-Actin Is Required for Proper Mouse Neural Crest Ontogeny

The mouse genome consists of six functional actin genes of which the expression patterns are temporally and spatially regulated during development and in the adult organism. Deletion of beta-actin in mouse is lethal during embryonic development, although there is compensatory expression of other actin isoforms. This suggests different isoform specific functions and, more in particular, an important function for beta-actin during early mammalian development. We here report a role for beta-actin during neural crest ontogeny. Although beta-actin null neural crest cells show expression of neural crest markers, less cells delaminate and their migration arrests shortly after. These phenotypes were associated with elevated apoptosis levels in neural crest cells, whereas proliferation levels were unchanged. Specifically the pre-migratory neural crest cells displayed higher levels of apoptosis, suggesting increased apoptosis in the neural tube accounts for the decreased amount of migrating neural crest cells seen in the beta-actin null embryos. These cells additionally displayed a lack of membrane bound N-cadherin and dramatic decrease in cadherin-11 expression which was more pronounced in the pre-migratory neural crest population, potentially indicating linkage between the cadherin-11 expression and apoptosis. By inhibiting ROCK ex vivo, the knockout neural crest cells regained migratory capacity and cadherin-11 expression was upregulated. We conclude that the presence of beta-actin is vital for survival, specifically of pre-migratory neural crest cells, their proper emigration from the neural tube and their subsequent migration. Furthermore, the absence of beta-actin affects cadherin-11 and N-cadherin function, which could partly be alleviated by ROCK inhibition, situating the Rho-ROCK signaling in a feedback loop with cadherin-11.

Embryonic rat vascular smooth muscle cells revisited - a model for neonatal, neointimal SMC or differentiated vascular stem cells?

BackgroundThe A10 and A7r5 cell lines derived from the thoracic aorta of embryonic rat are widely used as models of non-differentiated, neonatal and neointimal vascular smooth muscle cells in culture. The recent discovery of resident multipotent vascular stem cells within the vessel wall has necessitated the identity and origin of these vascular cells be revisited. In this context, we examined A10 and A7r5 cell lines to establish the similarities and differences between these cell lines and multipotent vascular stem cells isolated from adult rat aortas by determining their differentiation state, stem cell marker expression and their multipotency potential in vitro.MethodsVascular smooth muscle cell differentiation markers (alpha-actin, myosin heavy chain, calponin) and stem cell marker expression (Sox10, Sox17 and S100β) were assessed using immunocytochemistry, confocal microscopy, FACS analysis and real-time quantitative PCR.ResultsBoth A10 and A7r5 expressed vascular smooth muscle differentiation, markers, smooth muscle alpha - actin, smooth muscle myosin heavy chain and calponin. In parallel analysis, multipotent vascular stem cells isolated from rat aortic explants were immunocytochemically myosin heavy chain negative but positive for the neural stem cell markers Sox10+, a neural crest marker, Sox17+ the endoderm marker, and the glia marker, S100β+. This multipotent vascular stem cell marker profile was detected in both embryonic vascular cell lines in addition to the adventitial progenitor stem cell marker, stem cell antigen-1, Sca1+. Serum deprivation resulted in a significant increase in stem cell and smooth muscle cell differentiation marker expression, when compared to serum treated cells. Both cell types exhibited weak multipotency following adipocyte inductive stimulation. Moreover, Notch signaling blockade following γ-secretase inhibition with DAPT enhanced the expression of both vascular smooth muscle and stem cell markers.ConclusionsWe conclude that A10 and A7r5 cells share similar neural stem cell markers to both multipotent vascular stem cells and adventitial progenitors that are indicative of neointimal stem-derived smooth muscle cells. This may have important implications for their use in examining vascular contractile and proliferative phenotypes in vitro.

Human Embryonic Stem Cell-Derived Neural Crest Cells Capable of Expressing Markers of Osteochondral or Meningeal–Choroid Plexus Differentiation

The transcriptome and fate potential of three diverse human embryonic stem cell-derived clonal embryonic progenitor cell lines with markers of cephalic neural crest are compared when differentiated in the presence of combinations of TGFβ3, BMP4, SCF and HyStem-C matrices. The cell lines E69 and T42 were compared with MEL2, using gene expression microarrays, immunocytochemistry and ELISA. In the undifferentiated progenitor state, each line displayed unique markers of cranial neural crest including TFAP2A and CD24; however, none expressed distal HOX genes including HOXA2 or HOXB2, or the mesenchymal stem cell marker CD74. The lines also showed diverse responses when differentiated in the presence of exogenous BMP4, BMP4 and TGFβ3, SCF, and SCF and TGFβ3. The clones E69 and T42 showed a profound capacity for expression of endochondral ossification markers when differentiated in the presence of BMP4 and TGFβ3, choroid plexus markers in the presence of BMP4 alone, and leptomeningeal markers when differentiated in SCF without TGFβ3. The clones E69 and T42 may represent a scalable source of primitive cranial neural crest cells useful in the study of cranial embryology, and potentially cell-based therapy.

Abstract CN03-03: Use of the zebrafish for studying cancer genetics.

Abstract Melanoma is a devastating disease and there are few therapies that lead to substantial long-term remission for metastatic disease. We have created a zebrafish model of melanoma by overexpressing BRAF-V600E in the combination of P53 mutation. This fish develops melanoma at a characteristic rate and the tumors greatly resemble those of human melanomas. Examination of gene expression in the melanomas demonstrated neural crest transcription factor expression in the zebrafish melanomas. A neural crest marker called Crestin was substantially upregulated in the tumors compared to adjacent skin. A chemical screen was undertaken to find small molecules that prevent neural crest development during embryogenesis in the zebrafish, hoping to find a small molecule that would inhibit the gene expression signature in the tumor. A drug called leflunomide was found to inhibit transcription elongation in the crest and also in human melanomas. Combination of a BRAF inhibitor plus leflunomide led to a substantial decrease in human tumors when placed into nude mice. A Phase II clinical trial has been undertaken using the combination of BRAF inhibitor and leflunomide. Leflunomide has an important effect on transcription elongation that is dependent on a new tumor suppressor complex called the HEXIM complex. Inactivation of the HEXIM complex interferes with the effect of the drug. HEXIM has also been shown to be a tumor suppressor in our zebrafish model system. In an effort to understand the effects of leflunomide, we undertook a screen for a chemical suppressor of leflunomide. This demonstrated pathways that affected drug metabolism as well as transcription elongation. Our studies demonstrate a novel pathway for interrupting cancer development by blocking transcription elongation in combination with BRAF inhibitors. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):CN03-03. Citation Format: Leonard I. Zon. Use of the zebrafish for studying cancer genetics. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr CN03-03.

Human Adult Periodontal Ligament-Derived Cells Integrate and Differentiate after Implantation into the Adult Mammalian Brain

Previous studies suggest that neural crest (NC)-derived stem cells may reside in NC derivatives including the human periodontal ligament (hPDL). The isolation and manipulation of autologous NC-derived cells could be an accessible source of adult neural stem cells for their use in cell replacement and gene transfer to the diseased central nervous system. In this study, we examined the expression of NC markers and neural differentiation potential of hPDL-derived cells both in vitro and in vivo. In vitro we found that hPDL-derived cells expressed stem cell markers (Oct3/4, Nestin, Sox2, and Musashi-1) and a subset of NC cell markers (Slug, p75(NTR), Twist, and Sox9). hPDL-derived cells differentiated into neural-like cells based on cellular morphology and neural marker expression (TUJ1, MAP2, MAP1b, GAD65/67, GABA, NeuN, ChAT, GAT1, synaptophysin, GFAP, NG2, and O4). In vivo, hPDL-derived cells survive, migrate, and give rise to DCX(+), NF-M(+), GABA(+), GFAP(+), and NG2(+) cells after grafting the adult mouse brain. Some of the grafted hPDL-derived cells were located in stem cell niches such as the ventricular epithelium and the subventricular zone of the anterolateral ventricle wall as well as in the subgranular zone of the hippocampal dentate gyrus. Thus, the hPDL contains stem cells that originate from the NC and can differentiate into neural cells. The engraftment and differentiation properties of hPDL-derived cells in the adult brain indicate that they are a potential stem cell source to be used in neuroregenerative and/or neurotrophic medicine.

Role ofSp5as an essential early regulator of neural crest specification inxenopus

The neural crest (NC) is a multipotent embryonic cell population, which is induced by an integration of secreted signals including BMP, Wnt, and FGF and, subsequently, NC cell fates are specified by a regulatory network of specific transcription factors. This study was undertaken to identify a role of Sp5 transcription factor in vertebrates. Xenopus Sp5 is expressed in the prospective neural crest regions from gastrulation through the tadpole stages in early development. Knockdown of Sp5 caused severe defects in craniofacial cartilage, pigmentation, and dorsal fin. Gain- and loss-of-function of Sp5 led to up- and down-regulation of the expression of NC markers in the neural fold, respectively. In contrast, Sp5 had no effect on neural induction and patterning. Sp5 regulated the expression of neural plate border (NPB) specifiers, Msx1 and Pax3, and these regulatory factors recovered the expression of NC marker in the Sp5-deficient embryos. Depletion of Sp5 impaired NC induction by Wnt/β-catenin or FGF signal, whereas its co-expression rescued NC markers in embryos in which either signal was blocked. These results suggest that Sp5 functions as a critical early factor in the genetic cascade to regulate NC induction downstream of Wnt and FGF pathways.

Semicircular canal morphogenesis in the zebrafish inner ear requires the function of gpr126 (lauscher), an adhesion class G protein-coupled receptor gene.

Morphogenesis of the semicircular canal ducts in the vertebrate inner ear is a dramatic example of epithelial remodelling in the embryo, and failure of normal canal development results in vestibular dysfunction. In zebrafish and Xenopus, semicircular canal ducts develop when projections of epithelium, driven by extracellular matrix production, push into the otic vesicle and fuse to form pillars. We show that in the zebrafish, extracellular matrix gene expression is high during projection outgrowth and then rapidly downregulated after fusion. Enzymatic disruption of hyaluronan in the projections leads to their collapse and a failure to form pillars: as a result, the ears swell. We have cloned a zebrafish mutant, lauscher (lau), identified by its swollen ear phenotype. The primary defect in the ear is abnormal projection outgrowth and a failure of fusion to form the semicircular canal pillars. Otic expression of extracellular matrix components is highly disrupted: several genes fail to become downregulated and remain expressed at abnormally high levels into late larval stages. The lau mutations disrupt gpr126, an adhesion class G protein-coupled receptor gene. Expression of gpr126 is similar to that of sox10, an ear and neural crest marker, and is partially dependent on sox10 activity. Fusion of canal projections and downregulation of otic versican expression in a hypomorphic lau allele can be restored by cAMP agonists. We propose that Gpr126 acts through a cAMP-mediated pathway to control the outgrowth and adhesion of canal projections in the zebrafish ear via the regulation of extracellular matrix gene expression.

Late‐emigrating trunk neural crest cells in turtle embryos generate an osteogenic ectomesenchyme in the plastron

The turtle plastron is composed of a keratinized epidermis overlying nine dermal bones. Its developmental origin has been controversial; recent evidence suggests that the plastral bones derive from trunk neural crest cells (NCCs). This study extends the observations that there is a turtle-specific, second wave of trunk NCC delamination and migration, after the original NCCs have reached their destination and differentiated. This second wave was confirmed by immunohistochemistry in whole-mounts and serial sections, by injecting DiI (1,1', di-octadecyl-3,3,3',3',-tetramethylindo-carbocyanine perchlorate) into the lumen of the neural tube and tracing labeled cells into the plastron, and by isolating neural tubes from older turtle embryos and observing delaminating NCCs. This later migration gives rise to a plastral ectomesenchyme that expresses NCC markers and can be induced to initiate bone formation. The NCCs of this second migration have properties similar to those of the earlier NCCs, but also express markers characteristic of cranial NCCs. The majority of the cells of the plastron mesenchyme express neural crest markers, and have osteogenic differentiation capabilities that are similar or identical to craniofacial ectomesenchyme. Our evidence supports the contention that turtle plastron bones are derived from a late emigrating population of cells derived from the trunk neural crest.

Endolymphatic Periductal Channels: Possible Role in Ménière's Disease

Objectives: Characterize the osseous periductal channels surrounding the human endolymphatic duct. Endolymphatic hydrops is the fundamental pathophysiology underlying Ménière’s disease. Elucidating the regulation of endolymphatic circulation is central to understanding the cause of Ménière’s disease attacks of vertigo and hearing loss. Methods: Formalin-fixed, celloidin-embedded human temporal bones were sectioned to 20 microns, and every section was stained with hematoxylin & eosin. Amira software was used to construct a three dimensional image of the periductal channels. We performed immunohistochemical analysis using antibodies directed against vascular, lymphatic, and neural crest markers. The microanatomy was investigated with electron microscopy of the periductal channel epithelium. Finally, we injected horseradish peroxidase into the scala media of guinea pigs to investigate the flow patterns involving these peri-ductal channels. Results: Surrounding the human endolymphatic duct is a group of small channels that originate in the proximal cisternal area of the endolymphatic sac and terminate in the supporting tissue of the saccule, as demonstrated by 3D reconstructions. Endolymphatic hydrops was found in eight bones with blocked ducts and blocked or absent periductal channels. Proliferation of channels was evident in temporal bones when the channels were disrupted on one side. Immunohistochemical analysis results suggest a neuroectodermal origin, similar to the cells of the cochlear spiral ligament. Some contain micro-vessels with single endothelial walls that could represent lymphatics or empty capillaries while others suggest a complex absortive epithelium. Conclusions: This temporal bone study suggests a role for the periductal channels in absorption of endolymph.

Requirement for frzb and fzd7a in cranial neural crest convergence and extension mechanisms during zebrafish palate and jaw morphogenesis

Regulation of convergence and extension by wnt-frizzled signaling is a common theme in embryogenesis. This study examines the functional requirements of frzb and fzd7a in convergence and extension mechanisms during craniofacial development. Using a morpholino knockdown approach, we found that frzb and fzd7a are dispensable for directed migration of the bilateral trabeculae, but necessary for the convergence and extension of the palatal elements, where the extension process is mediated by chondrocyte proliferation, morphologic change and intercalation. In contrast, frzb and fzd7a are required for convergence of the mandibular prominences, where knockdown of either frzb or fzd7a resulted in complete loss of lower jaw structures. Further, we found that bapx1 was specifically downregulated in the wnt9a/frzb/fzd7a morphants, while general neural crest markers were unaffected. In addition, expression of wnt9a and frzb was also absent in the edn−/− mutant. Notably, over-expression of bapx1 was sufficient to partially rescue mandibular elements in the wnt9a/frzb/fzd7a morphants, demonstrating genetic epistasis of bapx1 acting downstream of edn1 and wnt9a/frzb/fzd7a in lower jaw development. This study underscores the important role of wnt-frizzled signaling in convergence and extension in palate and craniofacial morphogenesis, distinct regulation of upper vs. lower jaw structures, and integration of wnt-frizzled with endothelin signaling to coordinate shaping of the facial form.

Neural Crest Cells Isolated from the Bone Marrow of Transgenic Mice Express JCV T-Antigen

JC virus (JCV), a common human polyomavirus, is the etiological agent of the demyelinating disease, progressive multifocal leukoencephalopathy (PML). In addition to its role in PML, studies have demonstrated the transforming ability of the JCV early protein, T-antigen, and its association with some human cancers. JCV infection occurs in childhood and latent virus is thought to be maintained within the bone marrow, which harbors cells of hematopoietic and non-hematopoietic lineages. Here we show that non-hematopoietic mesenchymal stem cells (MSCs) isolated from the bone marrow of JCV T-antigen transgenic mice give rise to JCV T-antigen positive cells when cultured under neural conditions. JCV T-antigen positive cells exhibited neural crest characteristics and demonstrated p75, SOX-10 and nestin positivity. When cultured in conditions typical for mesenchymal cells, a population of T-antigen negative cells, which did not express neural crest markers arose from the MSCs. JCV T-antigen positive cells could be cultured long-term while maintaining their neural crest characteristics. When these cells were induced to differentiate into neural crest derivatives, JCV T-antigen was downregulated in cells differentiating into bone and maintained in glial cells expressing GFAP and S100. We conclude that JCV T-antigen can be stably expressed within a fraction of bone marrow cells differentiating along the neural crest/glial lineage when cultured in vitro. These findings identify a cell population within the bone marrow permissible for JCV early gene expression suggesting the possibility that these cells could support persistent viral infection and thus provide clues toward understanding the role of the bone marrow in JCV latency and reactivation. Further, our data provides an excellent experimental model system for studying the cell-type specificity of JCV T-antigen expression, the role of bone marrow-derived stem cells in the pathogenesis of JCV-related diseases and the opportunities for the use of this model in development of therapeutic strategies.

Eif3ba regulates cranial neural crest development by modulating p53 in zebrafish

Congenital diseases caused by abnormal development of the cranial neural crest usually present craniofacial malformations and heart defects while the precise mechanism is not fully understood. Here, we show that the zebrafish eif3ba mutant caused by pseudo-typed retrovirus insertion exhibited a similar phenotype due to the hypogenesis of cranial neural crest cells (NCCs). The derivatives of cranial NCCs, including the NCC-derived cell population of pharyngeal arches, craniofacial cartilage, pigment cells and the myocardium derived from cardiac NCCs, were affected in this mutant. The expression of several neural crest marker genes, including crestin, dlx2a and nrp2b, was specifically reduced in the cranial regions of the eif3ba mutant. Through fluorescence-tracing of the cranial NCC migration marker nrp2b, we observed reduced intensity of NCC-derived cells in the heart. In addition, p53 was markedly up-regulated in the eif3ba mutant embryos, which correlated with pronounced apoptosis in the cranial area as shown by TUNEL staining. These findings suggest a novel function of eif3ba during embryonic development and a novel level of regulation in the process of cranial NCC development, in addition to providing a potential animal model to mimic congenital diseases due to cranial NCC defects. Furthermore, we report the identification of a novel transgenic fish line Et(gata2a:EGFP)pku418 to trace the migration of cranial NCCs (including cardiac NCCs); this may serve as an invaluable tool for investigating the development and dynamics of cranial NCCs during zebrafish embryogenesis.

The role of SOX10 during enteric nervous system development

The SOX10 transcription factor is a characteristic marker for migratory multipotent neural crest (NC) progenitors as well as several of their differentiated derivatives. The involvement of SOX10 in Waardenburg–Hirschsprung disease (pigmentation defects, deafness and intestinal aganglionosis) and studies of mutant animal models have contributed significantly to the understanding of its function in neural crest cells (NCC) in general and in the melanocytes and enteric nervous system (ENS) in particular. Cell-based studies have further demonstrated the important roles of this transcription factor in maintaining the NC progenitor cell number and in determining glial cell fate. Phenotypic variability observed among patients presenting with SOX10 mutations is in agreement with molecular genetics and animal model studies, which revealed that SOX10 cooperates with different partner factors; a number of genetic modifiers of SOX10 have been identified. This study reviews the expression, regulation, and function of SOX10 in normal development of the ENS and in disease conditions, as well as the genetic and molecular interactions of SOX10 with other ENS genes/factors. We also discuss future research areas. Further understanding of SOX10 function will benefit from genomic and cell biological studies that integrate the cell-intrinsic molecular mechanisms and the interactions of the enteric NCC with the niche environment.

Development of the human aortic arch system captured in an interactive three‐dimensional reference model

Variations and mutations in the human genome, such as 22q11.2 microdeletion, can increase the risk for congenital defects, including aortic arch malformations. Animal models are increasingly expanding our molecular and genetic insights into aortic arch development. However, in order to justify animal-to-human extrapolations, a human morphological, and molecular reference model would be of great value, but is currently lacking. Here, we present interactive three-dimensional reconstructions of the developing human aortic arch system, supplemented with the protein distribution of developmental markers for patterning and growth, including T-box transcription factor TBX1, a major candidate for the phenotypes found in patients with the 22q11.2 microdeletion. These reconstructions and expression data facilitate unbiased interpretations, and reveal previously unappreciated aspects of human aortic arch development. Based on our reconstructions and on reported congenital anomalies of the pulmonary trunk and tributaries, we postulate that the pulmonary arteries originate from the aortic sac, rather than from the sixth pharyngeal arch arteries. Similar to mouse, TBX1 is expressed in pharyngeal mesenchyme and epithelia. The endothelium of the pharyngeal arch arteries is largely negative for TBX1 and family member TBX2 but expresses neural crest marker AP2α, which gradually decreases with ongoing development of vascular smooth muscle. At early stages, the pharyngeal arch arteries, aortic sac, and the dorsal aortae in particular were largely negative for proliferation marker Ki67, potentially an important parameter during aortic arch system remodeling. Together, our data support current animal-to-human extrapolations and future genetic and molecular analyses using animal models of congenital heart disease. © 2013 Wiley Periodicals, Inc.

Abstract 3744: G-CSF-dependent regulation of STAT3 in neuroblastoma cancer stem cell subpopulations.

Abstract Neuroblastoma is a neural crest derived malignancy arising form peripheral sympathetic nervous system of infants and young children. We have recently defined a highly tumorigenic sub-population of neuroblastoma cells based on surface expression of CD114 (the G-CSF receptor, CSF3R). These cells meet the operational criteria for cancer stem cells based on their self-renewal, differentiation and lineage restricted tumorigenicity, and can be isolated directly from primary tumor biopsy specimens. As STAT3 activation is a canonical downstream event of G-CSF receptor ligation, is critical for neuronal specification, and is implicated in cancer stem cell maintenance, we hypothesized that inhibition of the STAT3 pathway may functionally inhibit the cancer stem cell-like neuroblastoma subpopulation. We used pathway-specific qPCR arrays to evaluate differential gene expression in CD114+ and CD114- tumor subpopulations. As predicted, STAT3 target genes were up-regulated in the CD114+ stem cell-like population. In addition, critical genes controlling early neural crest specification, cell cycle progression, self-renewal, and maintenance of pluripotency were up-regulated in CD114+ population while markers of mature mesenchymal neural crest were increased in CD114- population. Treatment of Neuroblastoma cell lines with G-CSF increased both CD114 expression and STAT3 transcriptional activation in a dose and time dependent manner. Phospho-flow analysis confirmed STAT3 phosphorylation (Y705) in response to exogenous G-CSF. The addition of a STAT3 inhibitor which prevents phosphorylation and dimerization of STAT3 (Stattic), specifically abrogated the effect of exogenous G-CSF treatment. Finally, we identified putative STAT3 binding sites within the 5’UTR of the CSF3R gene and confirmed binding of STAT3 and pSTAT3 using chromatin immunoprecipitation analysis (ChIP-qPCR). We demonstrate significant enrichment of binding in response to exogenous G-CSF within CD114 receptor positive subpopulation in contrast to CD114- population. These data support a model for a positive-feedback loop where G-CSF triggers STAT3 dependent up-regulation of the G-CSF receptor, further sensitizing these cells to exogenous ligand, promoting STAT3 transcriptional activation, and contributing to stem cell maintenance. Our data support further evaluation of the potential clinical role for specific STAT3 inhibitors as anti-cancer stem cell therapy in neuroblastoma. Citation Format: Saurabh Agarwal, Zaowen Chen, Danielle Hsu, Eugene Kim, Jason M. Shohet. G-CSF-dependent regulation of STAT3 in neuroblastoma cancer stem cell subpopulations. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3744. doi:10.1158/1538-7445.AM2013-3744

Abstract 3234: Comparative oncogenomics: identifying melanoma drivers from zebrafish to humans.

Abstract The elucidation of core networks driving tumorigenesis is greatly enhanced by studying tumors from multiple species. A comparison of tumors between evolutionarily distant organisms such as humans and fish offers a particularly powerful method to identify such core functionalities. In the past several years, the zebrafish has emerged as an important vertebrate model of cancer because it develops tumors that are histologically and functionally similar to the human disease. Overexpression of the BRAFV600E oncogene under the melanocyte-specific mitf promoter leads to a 100% penetrant melanoma in the context of p53 loss of function. We have previously shown that these zebrafish tumors exhibit characteristics of neural crest progenitors, rather than differentiated melanocytes, similar to many human melanomas which also overexpress the neural crest markers sox10 and ednrb. To identify the genomic lesions that are common to both species, we are performing RNA and DNA-sequencing of the zebrafish tumors and comparing them to human melanoma resequencing samples to identify common driver events. To facilitate this, we have developed technologies for both short and long term culture of zebrafish melanoma cell lines, along with normal tissue from each individual animal. One such melanoma line, ZMEL1, has been comprehensively characterized. RNA-seq demonstrated upregulation of neural crest genes such as ednrb, and pathway analysis indicated enrichment for ERK/MAP kinase and PI3K/AKT signaling pathways, and these tumors are sensitive to inhibition by the BRAF inhibitor PLX4720. Whole-genome sequencing (WGS) was performed on tumor cell line and matched normal tissue to a depth of 39X coverage on the Illumina platform. A total of 5193 single basepair mutations were identified, of which 2% were found in the coding region, 41% in intronic regions and 56% in the intergenic DNA. Within the coding region, 25% were G>C and 18% T>C substitutions, with approximately equal number of non-synonymous to silent mutations. Coding mutations in several genes such as rps6kb1b and plb1 are of particular interest because of their reported mutation in human cancer as well. 35 regions of copy number changes were found, comprising over 600 genes, and these include amplification of known oncogenes such as SETDB1. Functional analysis of the genomic changes common to both zebrafish and human tumors is now being undertaken using in vitro and in vivo melanoma models. In summary, comparative oncogenomics between zebrafish and human melanoma provides a powerful method for filtering the vast number of changes down to a list of manageable, testable hypotheses. This method can be broadly applied to other tumors types. Citation Format: Richard M. White, Erin Langdon, Charles Kaufman, Ellen van Rooijen, Jennifer Yen, Peter Van Loo, Leonard Zon. Comparative oncogenomics: identifying melanoma drivers from zebrafish to humans. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3234. doi:10.1158/1538-7445.AM2013-3234 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Old before their time: the ancient origins of the neural crest.

Old before their time: the ancient origins of the neural crest.

Potential use of skin-derived precursors (SKPs) in establishing a cell-based treatment model for Hirschsprung's disease

BackgroundHirschsprung's (HSCR) disease is characterized by absence of ganglia in the distant bowel. Skin-derived precursor cells (SKPs) are somatic stem cells located in the bulge of hair follicles with high neural plasticity. In this study, we elucidated the therapeutic potential of SKPs for replenishing absent ganglia in HSCR bowel. MethodsSKPs were isolated from mouse or human skin and cultured in neural differentiation medium to generate various types of neural cells. Expression of stem cell and neural differentiation markers were monitored by reverse-transcription polymerase chain reaction and immunocytochemistry, respectively. Engraftment and differentiation potentials of SKPs were further assessed using ex vivo gut culture with Retk/k aganglionic gut. ResultsExpression studies revealed that SKPs express a panel of neural crest markers and three key stemness factors (Klf4, c-Myc and Sox2), which may account for the multipotency of these cells. Subsequent differentiation assays directly demonstrated that both mouse and human SKPs retain high differentiation capacities to form enteric neurons, and glia. Importantly, with ex vivo gut explants assay, we further showed that SKPs colonize and differentiate in the Retk/k aganglionic hindgut explants. ConclusionOur data suggest that SKPs may represent an alternative source of stem cells for the study of cell-based therapy for HSCR.

Intervertebral Disc-Derived Stem Cells

An in vitro and in vivo evaluation of intervertebral disc (IVD)-derived stem/progenitor cells. To determine the chondrogenic, adipogenic, osteogenic, and neurogenic differentiation capacity of disc-derived stem/progenitor cells in vitro and neurogenic differentiation in vivo. Tissue repair strategies require a source of appropriate cells that could be used to replace dead or damaged cells and tissues such as stem cells. Here we examined the potential use of IVD-derived stem cells in regenerative medicine approaches and neural repair. Nonchondrodystrophic canine IVD nucleus pulposus (NP) cells were used to generate stem/progenitor cells (NP progenitor cells [NPPCs]) and the NPPCs were differentiated in vitro into chondrogenic, adipogenic, and neurogenic lineages and in vivo into the neurogenic lineage. NPPCs were compared with bone marrow-derived mesenchymal (stromal) stem cells in terms of the expression of stemness genes. The expression of the neural crest marker protein 0 and the Brachyury gene were evaluated in NP cells and NPPCs. NPPCs contain stem/progenitor cells and express "stemness" genes such as Sox2, Oct3/4, Nanog, CD133, Nestin, and neural cell adhesion molecule but differ from mesenchymal (stromal) stem cells in the higher expression of the Nanog gene by NPPCs. NPPCs do not express protein 0 or the Brachyury gene both of which are expressed by the totality of IVD NP cells. The percentage of NPPCs within the IVD is 1% of the total as derived by colony-forming assay. NPPCs are capable of differentiating along chondrogenic, adipogenic, and neurogenic lineages in vitro and into oligodendrocyte, neuron, and astroglial specific precursor cells in vivo within the compact myelin-deficient shiverer mouse. We propose that the IVD NP represents a regenerative niche suggesting that the IVD could represent a readily accessible source of precursor cells for neural repair and regeneration.