Trunk Neural Crest Cells Research Articles

Surgical histopathology of limited dorsal myeloschisis with flat skin lesion.

Limited dorsal myeloschisis (LDM) is characterized by two invariable features: a focal closed neural tube defect and a fibroneural stalk linking the skin lesion to the underlying spinal cord. Although detailed histopathological findings of the LDM stalk were originally described by Pang et al., the precise relationship between the histopathological findings and clinical manifestations including intraoperative findings has not been fully determined. We retrospectively analyzed the histopathological findings of the almost entire stalk and their relevance to the clinical manifestations in six Japanese LDM patients with flat skin lesions. Glial fibrillary acidic protein (GFAP)-immunopositive neuroglial tissues were observed in three of the six patients. Unlike neuroglial tissues, peripheral nerve fibers were observed in every stalk. In four patients, dermal melanocytosis, "Mongolian spot," was seen surrounding the cigarette-burn lesion. In three of these four patients, numerous melanocytes were distributed linearly along the long axis of the LDM stalk, which might represent migration of melanocytes from trunk neural crest cells during formation of the LDM stalk. Immunopositivity for GFAP in the LDM stalk was observed in as few as 50% of our patients, despite the relatively extensive histopathological examination. We confirm that the clinical diagnosis of LDM should be made based on comprehensive histopathological examination as well as clinical manifestations. The profuse network of peripheral nerve fibers in every stalk and the high incidence of melanocyte accumulation associated with dermal melanocytosis might assist the histopathological diagnosis of LDM.

Neural Crest Transplantation Reveals Key Roles in the Evolution of Cavefish Development.

Evolutionary changes in Astyanax mexicanus cavefish with respect to conspecific surface fish, including the regression of eyes, loss of pigmentation, and modification of the cranial skeleton, involve derivatives of the neural crest. However, the role of neural crest cells in cavefish evolution and development is poorly understood. One of the reasons is that experimental methods for neural crest analysis are not well developed in the Astyanax system. Here we describe neural crest transplantation between Astyanax surface fish and cavefish embryos. We found differences in the migration of cranial neural crest cells transplanted from the surface fish anterior hindbrain to the same region of surface fish or cavefish hosts. Cranial neural crest cells migrated extensively throughout the head, and to a lesser extent the trunk, in surface fish hosts but their migration was mostly restricted to the anterior and dorsal head regions in cavefish hosts. Cranial neural crest cells derived from the surface fish transplants invaded the degenerating eyes of cavefish hosts, resulting in increased eye size and suggesting that cavefish neural crest cells are defective in forming optic derivatives. We found that melanophores were formed in albino cavefish from grafts of surface fish trunk neural crest cells, showing that the cavefish tissue environment is conducive for pigment cell development, and implicating intrinsic changes in cavefish neural crest cells in loss of body pigmentation. It is concluded that changes in neural crest cells play key roles in the evolution of cavefish development.

The convergent roles of CD271/p75 in neural crest-derived melanoma plasticity

The embryonic microenvironment is an important source of signals that promote multipotent cells to adopt a specific fate and direct cells along distinct migratory pathways. Yet, the ability of the embryonic microenvironment to retain multipotent progenitors or reprogram de-differentiated cells is less clear. Mistakes in cell differentiation or migration often result in developmental defects and tumorigenesis, including aggressive cancers that share many characteristics with embryonic progenitor cells. This is a striking feature of the vertebrate neural crest, a multipotent and highly migratory cell population first identified by His (1868) with the potential to metamorphose into aggressive melanoma cancer. In this perspective, we address the roles of CD271/p75 in tumor initiation, phenotype switching and reprogramming of metastatic melanoma and discuss the convergence of these roles in melanoma plasticity.

Late‐Migrating Neural Crest Cells in Turtle Embryos Display Strong Osteogenic Potential

Turtle plastron bones develop by intramembranous ossification, suggesting that they are derived, like the facial bones, from neural crest cells. Using cell‐labeling and neural tube explant cultures, we have shown that cells expressing neural crest markers emerge from the trunk neural tube in the turtle Trachemys scripta in two migratory phases over a greatly extended period. These findings were confirmed by electroporation of plasmids driving expression of fluorescent proteins. The early phase of migration (comparable to the period of neural crest cell (NCC) migration in other amniotes) extends until stage G10–11. These cells give rise to typical NCC derivatives. The NCCs that emerge late (beginning in stage G15–16 turtle embryos, well beyond the stage of neural crest migration in chick or mouse embryos) appear to migrate ventrally to form an ectomesenchymal dermis that gives rise to the bones of the plastron. Thus, there appear to be two distinct migratory phases in vivo. To test the hypothesis that the second wave of trunk NCCs in turtle embryos is capable of differentiating into bone, T. scripta neural tubes were cultured. The resulting NCCs were allowed to differentiate, and the differentiated cell types produced were analyzed by immunofluorescence. A subset of the cultures produced melanocytes, a typical trunk NCC‐derived cell type, while virtually all of the cultures produced a substantial number of osteoblasts. Our results suggest that the late trunk NCCs are predisposed to differentiate into osteoblasts, and thus provide good candidates for the cells that form the plastron. Craniosynostosis is a common human developmental deformity involving premature fusion of the calvarial sutures between the bones of the skull. A better understanding of intramembranous ossification, and analysis of an enriched population of osteogenic NCCs, could result in improved treatment options.Support or Funding InformationNational Science Foundation, Millersville University, and Stowers InstituteThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Neuregulin‐1 is a chemoattractant and chemokinetic molecule for trunk neural crest cells

Trunk neural crest cells migrate rapidly along characteristic pathways within the developing vertebrate embryo. Proper trunk neural crest cell migration is necessary for the morphogenesis of much of the peripheral nervous system, melanocytes, and the adrenal medulla. Numerous molecules help guide trunk neural crest cell migration throughout the early embryo. The trophic factor NRG1 is a chemoattractant through in vitro chemotaxis assays and in vivo silencing via a DN-erbB receptor. Interestingly, we also observed changes in migratory responses consistent with a chemokinetic effect of NRG1 in trunk neural crest velocity. NRG1 is a trunk neural crest cell chemoattractant and chemokinetic molecule. Developmental Dynamics 247:888-902, 2018.. © 2018 Wiley Periodicals, Inc.

Establishment of a murine culture system for modeling the temporal progression of cranial and trunk neural crest cell differentiation.

ABSTRACTThe neural crest (NC) is a transient population of embryonic progenitors that are implicated in a diverse range of congenital birth defects and pediatric syndromes. The broad spectrum of NC-related disorders can be attributed to the wide variety of differentiated cell types arising from the NC. In vitro models of NC development provide a powerful platform for testing the relative contributions of intrinsic and extrinsic factors mediating NC differentiation under normal and pathogenic conditions. Although differentiation is a dynamic process that unfolds over time, currently, there is no well-defined chronology that characterizes the in vitro progression of NC differentiation towards specific cell fates. In this study, we have optimized culture conditions for expansion of primary murine NC cells that give rise to both ectodermal and mesoectodermal derivatives, even after multiple passages. Significantly, we have delineated highly reproducible timelines that include distinct intermediate stages for lineage-specific NC differentiation in vitro. In addition, isolating both cranial and trunk NC cells from the same embryos enabled us to make direct comparisons between the two cell populations over the course of differentiation. Our results define characteristic changes in cell morphology and behavior that track the temporal progression of NC cells as they differentiate along the neuronal, glial and chondrogenic lineages in vitro. These benchmarks constitute a chronological baseline for assessing how genetic or environmental disruptions may facilitate or impede NC differentiation. Introducing a temporal dimension substantially increases the power of this platform for screening drugs or chemicals for developmental toxicity or therapeutic potential. This article has an associated First Person interview with the first author of the paper.

Trunk neural crest origin of dermal denticles in a cartilaginous fish

Cartilaginous fishes (e.g., sharks and skates) possess a postcranial dermal skeleton consisting of tooth-like "denticles" embedded within their skin. As with teeth, the principal skeletal tissue of dermal denticles is dentine. In the head, cranial neural crest cells give rise to the dentine-producing cells (odontoblasts) of teeth. However, trunk neural crest cells are generally regarded as nonskeletogenic, and so the embryonic origin of trunk denticle odontoblasts remains unresolved. Here, we use expression of FoxD3 to pinpoint the specification and emigration of trunk neural crest cells in embryos of a cartilaginous fish, the little skate (Leucoraja erinacea). Using cell lineage tracing, we further demonstrate that trunk neural crest cells do, in fact, give rise to odontoblasts of trunk dermal denticles. These findings expand the repertoire of vertebrate trunk neural crest cell fates during normal development, highlight the likely primitive skeletogenic potential of this cell population, and point to a neural crest origin of dentine throughout the ancestral vertebrate dermal skeleton.

TMOD-09. HUMAN PLURIPOTENT STEM CELL-BASED MODELS OF NEUROBLASTOMA

Neuroblastoma (NB) is a malignant tumor that accounts for ~15% of all pediatric cancer mortality. Nearly half of patients present with high-risk disease that has poor outcome, and the proto-oncogene MYCN is amplified in 45% of high-risk cases. Further, relapsed neuroblastoma demonstrates frequent activation of mitogen activated protein kinase (MAPK) signaling, including mutations in tumor suppressor NF1 and epigenetic silencing of the gene. Human induced pluripotent stem cells (iPSCs) represent a valuable tool by which to further dissect the genetic requirements of high-risk NB. Human iPSCs are superior to genetically engineered mouse models (GEMMs) at capturing the telomere biology and chromosomal landscape of primary human neuroblastoma. Further, they can be genetically manipulated to determine the genetic drivers of the disease. As proof of principle, we first differentiated normal human iPSCs toward trunk neural crest cells (NCC), the putative cells of origin for NB. We subsequently introduced established genetic drivers of NB (MYCN, ALK) and orthotopically implanted the resulting trunk NCCs into the renal capsules of immunocompromised mice. Three months post injection, tumors developed in 60% of MYCN/ALK mice, 10% in MYCN alone, and no tumors developed in ALK alone or empty vector. Tumors were transplantable and expressed markers typically found in NB, while lacking markers of other tumors such as rhabdomyosarcoma, Ewing sarcoma, and lymphoma. To show we can use our model to evaluate candidate drivers of NB, we knocked out NF1 in MYCN trunk NCCs using CRISPR/Cas9 and implanted these cells orthotopically in mice. 80% of mice with MYCN/NF1 modified cells developed tumors while NF1 knockout alone did not. Thus, we have generated the first human iPSC model of NB driven by MYCN and have shown the utility of our model in validating cooperating mutations. MH and TZ contributed equally to this work.

Asymmetric localization of DLC1 defines avian trunk neural crest polarity for directional delamination and migration

Following epithelial-mesenchymal transition, acquisition of avian trunk neural crest cell (NCC) polarity is prerequisite for directional delamination and migration, which in turn is essential for peripheral nervous system development. However, how this cell polarization is established and regulated remains unknown. Here we demonstrate that, using the RHOA biosensor in vivo and in vitro, the initiation of NCC polarization is accompanied by highly activated RHOA in the cytoplasm at the cell rear and its fluctuating activity at the front edge. This differential RHOA activity determines polarized NC morphology and motility, and is regulated by the asymmetrically localized RhoGAP Deleted in liver cancer (DLC1) in the cytoplasm at the cell front. Importantly, the association of DLC1 with NEDD9 is crucial for its asymmetric localization and differential RHOA activity. Moreover, NC specifiers, SOX9 and SOX10, regulate NEDD9 and DLC1 expression, respectively. These results present a SOX9/SOX10-NEDD9/DLC1-RHOA regulatory axis to govern NCC migratory polarization.

Atg7-Mediated Autophagy Is Involved in the Neural Crest Cell Generation in Chick Embryo.

Autophagy plays a very important role in numerous physiological and pathological events. However, it still remains unclear whether Atg7-induced autophagy is involved in the regulation of neural crest cell production. In this study, we found the co-location of Atg7 and Pax7+ neural crest cells in early chick embryo development. Upregulation of Atg7 with unilateral transfection of full-length Atg7 increased Pax7+ and HNK-1+ cephalic and trunk neural crest cell numbers compared to either Control-GFP transfection or opposite neural tubes, suggesting that Atg7 over-expression in neural tubes could enhance the production of neural crest cells. BMP4 in situ hybridization and p-Smad1/5/8 immunofluorescent staining demonstrated that upregulation of Atg7 in neural tubes suppressed the BMP4/Smad signaling, which is considered to promote the delamination of neural crest cells. Interestingly, upregulation of Atg7 in neural tubes could significantly accelerate cell progression into the S phase, implying that Atg7 modulates cell cycle progression. However, β-catenin expression was not significantly altered. Finally, we demonstrated that upregulation of the Atg7 gene could activate autophagy as did Atg8. We have also observed that similar phenotypes, such as more HNK-1+ neural crest cells in the unilateral Atg8 transfection side of neural tubes, and the transfection with full-length Atg8-GFP certainly promote the numbers of BrdU+ neural crest cells in comparison to the GFP control. Taken together, we reveal that Atg7-induced autophagy is involved in regulating the production of neural crest cells in early chick embryos through the modification of the cell cycle.

Trunk neural crest cells: formation, migration and beyond.

Neural crest cells (NCCs) are a multipotent, migratory cell population that generates an astonishingly diverse array of cell types during vertebrate development. The trunk neural crest has long been considered of particular significance. First, it has been held that the trunk neural crest has a morphogenetic role, acting to coordinate the development of the peripheral nervous system, secretory cells of the endocrine system and pigment cells of the skin. Second, the trunk neural crest additionally has skeletal potential. However, it has been demonstrated that a key role of the trunk neural crest streams is to organize the innervation of the intestine. Although trunk NCCs have a limited capacity for self-renewal, sometimes they become neural-crest-derived tumor cells and reveal the fact that that NCCs and tumor cells share the same molecular machinery. In this review we describe the routes taken by trunk NCCs and consider the signals and cues that pattern these trajectories. We also discuss recent advances in the characterization of the properties of trunk NCCs for various model organisms in order to highlight common themes. Finally, looking to the future, we discuss the need to translate the wealth of data from animal studies to the clinical area in order to develop treatments for neural crest-related human diseases.

A potential inhibitory function of draxin in regulating mouse trunk neural crest migration

Draxin is a repulsive axon guidance protein that plays important roles in the formation of three commissures in the central nervous system and dorsal interneuron 3 (dI3) in the chick spinal cord. In the present study, we report the expression pattern of mouse draxin in the embryonic mouse trunk spinal cord. In the presence of draxin, the longest net migration length of a migrating mouse trunk neural crest cell was significantly reduced. In addition, the relative number of apolar neural crest cells increased as the draxin treatment time increased. Draxin caused actin cytoskeleton rearrangement in the migrating trunk neural crest cells. Our data suggest that draxin may regulate mouse trunk neural crest cell migration by the rearrangement of cell actin cytoskeleton and by reducing the polarization activity of these cells subsequently.

Reprogramming of avian neural crest axial identity and cell fate.

Neural crest populations along the embryonic body axis of vertebrates differ in developmental potential and fate, so that only the cranial neural crest can contribute to the craniofacial skeleton in vivo. We explored the regulatory program that imbues the cranial crest with its specialized features. Using axial-level specific enhancers to isolate and perform genome-wide profiling of the cranial versus trunk neural crest in chick embryos, we identified and characterized regulatory relationships between a set of cranial-specific transcription factors. Introducing components of this circuit into neural crest cells of the trunk alters their identity and endows these cells with the ability to give rise to chondroblasts in vivo. Our results demonstrate that gene regulatory circuits that support the formation of particular neural crest derivatives may be used to reprogram specific neural crest-derived cell types.

Leader Cells Define Directionality of Trunk, but Not Cranial, Neural Crest Cell Migration.

SummaryCollective cell migration is fundamental for life and a hallmark of cancer. Neural crest (NC) cells migrate collectively, but the mechanisms governing this process remain controversial. Previous analyses in Xenopus indicate that cranial NC (CNC) cells are a homogeneous population relying on cell-cell interactions for directional migration, while chick embryo analyses suggest a heterogeneous population with leader cells instructing directionality. Our data in chick and zebrafish embryos show that CNC cells do not require leader cells for migration and all cells present similar migratory capacities. In contrast, laser ablation of trunk NC (TNC) cells shows that leader cells direct movement and cell-cell contacts are required for migration. Moreover, leader and follower identities are acquired before the initiation of migration and remain fixed thereafter. Thus, two distinct mechanisms establish the directionality of CNC cells and TNC cells. This implies the existence of multiple molecular mechanisms for collective cell migration.

Bone morphogenetic protein 4 promotes craniofacial neural crest induction from human pluripotent stem cells.

Neural crest (NC) cells are a group of cells located in the neural folds at the boundary between the neural and epidermal ectoderm. Cranial NC cells migrate to the branchial arches and give rise to the majority of the craniofacial region, whereas trunk and tail NC cells contribute to the heart, enteric ganglia of the gut, melanocytes, sympathetic ganglia, and adrenal chromaffin cells. Positional information is indispensable for the regulation of cranial or trunk and tail NC cells. However, the mechanisms underlying the regulation of positional information during human NC induction have yet to be fully elucidated. In the present study, supplementation of bone morphogenetic protein (BMP) 4 in defined serum-free culture conditions including fibroblast growth factor-2 and Wnt3a from day 8 after NC specification induced the expression of cranial NC markers, AP2alpha, MSX1, and DLX1, during NC cell differentiation from human pluripotent stem cells. On the other hand, the proportion of cells expressing p75(NTR) or HNK1 decreased compared with that of cells cultured without BMP4, whereas gene expression analysis demonstrated that the expression levels of cranial NC-associated genes increased in BMP4-treated NC cells. These BMP4-treated NC cells were capable of differentiation into osteocytes and chondrocytes. The results of the present study indicate that BMP4 regulates cranial positioning during NC development.

Identification of the neural crest-specific enhancer of Seraf gene in avian peripheral nervous system development

In vertebrate embryos, trunk neural crest cells give rise to Schwann cells, along with other derivatives. In this study, to elucidate the molecular mechanisms for the Schwann cell specification, we aimed to identify enhancer elements responsible for the expression of the Seraf gene, the earliest marker for the Schwann cell precursors in the avian embryos. We first compared the genomic structure around the Seraf locus in various vertebrates, and found that, while mammals do not have a Seraf homolog, teleost fish species have it. However, the intergenic sequences around the Seraf locus are not conserved between zebrafish and chicken, consistent with the fact that fish Seraf expression is not Schwann cell precursor-specific. We thus compared the intergenic sequences around the Seraf locus among avian species, and identified a potential enhancer containing a cluster of Sox10-binding sites. Accordingly, the identified enhancer is activated in a neural crest-specific manner in transfected quail embryos. We also found that Sox10 activated the enhancer in cultured cells. Thus, our results revealed a new role of Sox10 in the earliest phase of the Schwann cell fate specification.

Neural crest defects in ephrin-B2 mutant mice are non-autonomous and originate from defects in the vasculature

Ephrin-B2, a member of the Eph/ephrin family of cell signaling molecules, has been implicated in the guidance of cranial and trunk neural crest cells (NCC) and development of the branchial arches(BA), but detailed examination in mice has been hindered by embryonic lethality of Efnb2 null loss of function due to a requirement in angiogenic remodeling. To elucidate the developmental roles for Efnb2, we generated a conditional rescue knock-in allele that allows rescue of ephrin-B2 specifically in the vascular endothelium (VE), but is otherwise ephrin-B2 deficient. Restoration of ephrin-B2 expression specifically to the VE completely circumvents angiogenic phenotypes, indicating that the requirement of ephrin-B2 in angiogenesis is limited to the VE. Surprisingly, we find that expression of ephrin-B2 specifically in the VE is also sufficient for normal NCC migration and that conversely, embryos in which ephrin-B2 is absent specifically from the VE exhibit NCC migration and survival defects. Disruption of vascular development independent of loss of ephrin-B2 function also leads to defects in NCC and BA development. Together, these data indicate that direct ephrin-B2 signaling to NCCs is not required for NCC guidance, which instead depends on proper organization of the embryonic vasculature.

Cdon promotes neural crest migration by regulating N-cadherin localization

Neural crest cells (NCCs) are essential embryonic progenitor cells that are unique to vertebrates and form a remarkably complex and coordinated system of highly motile cells. Migration of NCCs occurs along specific pathways within the embryo in response to both environmental cues and cell–cell interactions within the neural crest population. Here, we demonstrate a novel role for the putative Sonic hedgehog (Shh) receptor and cell adhesion regulator, cdon, in zebrafish neural crest migration. cdon is expressed in developing premigratory NCCs but is downregulated once the cells become migratory. Knockdown of cdon results in aberrant migration of trunk NCCs: crestin positive cells can emigrate out of the neural tube but stall shortly after the initiation of migration. Live cell imaging analysis demonstrates reduced directedness of migration, increased velocity and mispositioned cell protrusions. In addition, transplantation analysis suggests that cdon is required cell-autonomously for directed NCC migration in the trunk. Interestingly, N-cadherin is mislocalized following cdon knockdown suggesting that the role of cdon in NCCs is to regulate N-cadherin localization. Our results reveal a novel role for cdon in zebrafish neural crest migration, and suggest a mechanism by which Cdon is required to localize N-cadherin to the cell membrane in migratory NCCs for directed migration.

Mesodermal origin of median fin mesenchyme and tail muscle in amphibian larvae.

Mesenchyme is an embryonic precursor tissue that generates a range of structures in vertebrates including cartilage, bone, muscle, kidney, and the erythropoietic system. Mesenchyme originates from both mesoderm and the neural crest, an ectodermal cell population, via an epithelial to mesenchymal transition (EMT). Because ectodermal and mesodermal mesenchyme can form in close proximity and give rise to similar derivatives, the embryonic origin of many mesenchyme-derived tissues is still unclear. Recent work using genetic lineage tracing methods have upended classical ideas about the contributions of mesodermal mesenchyme and neural crest to particular structures. Using similar strategies in the Mexican axolotl (Ambystoma mexicanum), and the South African clawed toad (Xenopus laevis), we traced the origins of fin mesenchyme and tail muscle in amphibians. Here we present evidence that fin mesenchyme and striated tail muscle in both animals are derived solely from mesoderm and not from neural crest. In the context of recent work in zebrafish, our experiments suggest that trunk neural crest cells in the last common ancestor of tetrapods and ray-finned fish lacked the ability to form ectomesenchyme and its derivatives.

Chicken trunk neural crest migration visualized with HNK1

The development of the nervous system involves cells remaining within the neural tube (CNS) and a group of cells that delaminate from the dorsal neural tube and migrate extensively throughout the developing embryo called neural crest cells (NCC). These cells are a mesenchymal highly migratory group of cells that give rise to a wide variety of cell derivatives: melanocytes, sensory neurons, bone, Schwann cells, etc. But not all NCC can give rise to all derivatives, they have fate restrictions based on their axial level of origin: cranial, vagal, trunk and sacral. Our aim was to provide a thorough presentation on how does trunk neural crest cell migration looks in the chicken embryo, in wholemount and in sections using the unique chicken marker HNK1. The description presented here makes a good guideline for those interested in viewing trunk NCC migration patterns. We show how before HH14 there are few trunk NCC delaminating and migrating, but between HH15 through HH19 trunk NCC delaminate in large numbers. Melanocytes precursors begin to enter the dorsolateral pathway by HH17. We found that by HH20 HNK1 is not a valid good marker for NCC and that HNK1 is a better marker than Sox10 when looking at neural crest cells morphology and migration details.