Neurotrophic factor NT-3 displays a non-canonical cell guidance signaling function for cephalic neural crest cells

Chemotactic cell migration is triggered by extracellular concentration gradients of molecules segregated by target fields. Neural crest cells (NCCs), paradigmatic as an accurately moving cell population, undergo wide dispersion along multiple pathways, invading with precision defined sites of the embryo to differentiate into many derivatives. This report addresses the involvement of NT-3 in early colonization by cephalic NCCs invading the optic vesicle region. The results of in vitro and in vivo approaches showed that NCCs migrate directionally up an NT-3 concentration gradient. We also demonstrated the expression of NT-3 in the ocular region as well as their functional TrkB, TrkC and p75 receptors on cephalic NCCs. On whole-mount embryo, a perturbed distribution of NCCs colonizing the optic vesicle target field was shown after morpholino cancelation of cephalic NT-3 or TrkC receptor on NCCs, as well as in situ blocking of TrkC receptor of mesencephalic NCCs by specific antibody released from inserted microbeads. The present results strongly suggest that, among other complementary cell guidance factor(s), the chemotactic response of NCCs toward the ocular region NT-3 gradient is essential for spatiotemporal cell orientation, amplifying the functional scope of this neurotrophic factor as a molecular guide for the embryo cells, besides its well-known canonical functions.     

Insights into neural stem cell biology from flies

Drosophila neuroblasts are similar to mammalian neural stem cells in their ability to self-renew and to produce many different types of neurons and glial cells. In the past two decades, great advances have been made in understanding the molecular mechanisms underlying embryonic neuroblast formation, the establishment of cell polarity and the temporal regulation of cell fate. It is now a challenge to connect, at the molecular level, the different cell biological events underlying the transition from neural stem cell maintenance to differentiation. Progress has also been made in understanding the later stages of development, when neuroblasts become mitotically inactive, or quiescent, and are then reactivated postembryonically to generate the neurons that make up the adult nervous system. The ability to manipulate the steps leading from quiescence to proliferation and from proliferation to differentiation will have a major impact on the treatment of neurological injury and neurodegenerative disease.     

Chemotaxis of capacitated rabbit spermatozoa to follicular fluid revealed by a novel directionality-based assay

Precontact communication between gametes is established by chemotaxis. Sperm chemotaxis toward factor(s) in follicular fluid (FF) has been demonstrated in humans and mice. In humans, the chemotactic responsiveness is restricted to capacitated spermatozoa. Here, we investigated whether sperm chemotaxis to factor(s) present in FF also occurs in rabbits and, if so, whether only capacitated spermatozoa are chemotactically responsive. Chemotaxis assays were performed by videomicroscopy in a Zigmond chamber. We measured chemotactic responsiveness as a function of FF dilution by means of a novel directionality-based method that considers the ratio between the distances traveled by the spermatozoa both parallel to the chemoattractant gradient and perpendicular to it. A peak of maximal response was observed at 10(-4) dilution of FF, resulting in a typical chemotactic concentration-dependent curve in which 23% of the spermatozoa were chemotactically responsive. In contrast, the percentage of cells exhibiting FF-dependent enhanced speed of swimming increased with the FF concentration, whereas the percentage of cells maintaining linear motility decreased with the FF concentration. The percentages of chemotactically responsive cells were very similar to those of capacitated spermatozoa. Depletion of the latter by stimulation of the acrosome reaction resulted in a total loss of the chemotactic response, whereas the reappearance of capacitated cells resulted in a recovery of chemotactic responsiveness. We conclude that rabbit spermatozoa, like human spermatozoa, are chemotactically responsive to FF factor(s) and acquire this responsiveness as part of the capacitation process.     

The stem cells of the neural crest

In the vertebrate embryo, the neurectodermal neural crest cells (NCC) have remarkably broad potencies, giving rise, after a migratory phase, to neurons and glial cells in the peripheral nervous system, and to skin melanocytes, being all designated here as “neural” derivatives. NC-derived cells also include non-neural, “mesenchymal” cell types like chondrocytes and bone cells, myofibroblasts and adipocytes, which largely contribute to the head structures in amniotes. Similar to the blood cell system, the NC is therefore a valuable model to investigate the mechanisms of cell lineage diversification in vertebrates. Whether NCC are endowed with multiple differentiation potentials or if, conversely, they are a mosaic of different committed cells is an important ongoing issue to understand the ontogeny of NC derivatives in normal development and pathological conditions. Here we focus on recent findings that established the presence in the early migratory NC of the avian embryo, of a multipotent progenitor endowed with both mesenchymal and neural differentiation capacities. This “mesenchymal-neural” clonogenic cell lies upstream of all the other NC progenitors known so far and shows increased frequency when single cell cultures are treated with the Sonic Hedgehog signaling molecule. These findings are discussed in the context of the broad potentials of NC stem cells recently evidenced in certain adult mammalian tissues.     

FGF8 signaling is chemotactic for cardiac neural crest cells

Cardiac neural crest cells migrate into the pharyngeal arches where they support development of the pharyngeal arch arteries. The pharyngeal endoderm and ectoderm both express high levels of FGF8. We hypothesized that FGF8 is chemotactic for cardiac crest cells. To begin testing this hypothesis, cardiac crest was explanted for migration assays under various conditions. Cardiac neural crest cells migrated more in response to FGF8. Single cell tracing indicated that this was not due to proliferation and subsequent transwell assays showed that the cells migrate toward an FGF8 source. The migratory response was mediated by FGF receptors (FGFR) 1 and 3 and MAPK/ERK intracellular signaling. To test whether FGF8 is chemokinetic and/or chemotactic in vivo, dominant negative FGFR1 was electroporated into the premigratory cardiac neural crest. Cells expressing the dominant negative receptor migrated slower than normal cardiac neural crest cells and were prone to remain in the vicinity of the neural tube and die. Treating with the FGFR1 inhibitor, SU5402 or an FGFR3 function-blocking antibody also slowed neural crest migration. FGF8 over-signaling enhanced neural crest migration. Neural crest cells migrated to an FGF8-soaked bead placed dorsal to the pharynx. Finally, an FGF8 producing plasmid was electroporated into an ectopic site in the ventral pharyngeal endoderm. The FGF8 producing cells attracted a thick layer of mesenchymal cells. DiI labeling of the neural crest as well as quail-to-chick neural crest chimeras showed that neural crest cells migrated to and around the ectopic site of FGF8 expression. These results showing that FGF8 is chemotactic and chemokinetic for cardiac neural crest adds another dimension to understanding the relationship of FGF8 and cardiac neural crest in cardiovascular defects.     

Taking neural crest stem cells to new heights

The carotid body is an organ of the peripheral nervous system that senses oxygen concentration in the blood and responds to changes by regulating breathing. Pardal et al. (2007) now report the discovery of carotid body stem cells, which proliferate in response to hypoxia and generate neurons that secrete dopamine. This new source of adult stem cells may be useful in therapies for treating Parkinson's disease.     

Insights from a sea lamprey into the evolution of neural crest gene regulatory network

The neural crest is a vertebrate innovation that forms at the embryonic neural plate border, transforms from epithelial to mesenchymal, migrates extensively throughout the embryo along well-defined pathways, and differentiates into a plethora of derivatives that include elements of peripheral nervous system, craniofacial skeleton, melanocytes, etc. The complex process of neural crest formation is guided by multiple regulatory modules that define neural crest gene regulatory network (NC GRN), which allows the neural crest to progressively acquire all of its defining characteristics. The molecular study of neural crest formation in lamprey, a basal extant vertebrate, consisting in identification and functional tests of molecular elements at each regulatory level of this network, has helped address the question of the timing of emergence of NC GRN and define its basal state. The results have revealed striking conservation in deployment of upstream factors and regulatory modules, suggesting that proximal portions of the network arose early in vertebrate evolution and have been tightly conserved for more than 500 million years. In contrast, certain differences were observed in deployment of some neural crest specifier and downstream effector genes expected to confer species-specific migratory and differentiation properties.     

Building stable chains with motile agents: Insights into the morphology of enteric neural crest cell migration

A defining characteristic of the normal development of the enteric nervous system (ENS) is the existence of an enteric neural crest (ENC) cell colonization wave, where the ENC cells form stable chains often associated with axons and near the vascular network. However, within this evolving neural network, the individual ENC cell elements constantly move, change direction and appear to act independently of neighbors. Three possible hypotheses are investigated. The simplest of these postulates that the ENS follows the vascular network as a template. We present evidence which does not support this hypothesis. Two viable alternatives are either that (i) the axons muster the ENC cells, providing the pattern for the chain migration or (ii) ENC cells form chains and the axons follow these paths. These two hypotheses are explored by developing a stochastic cellular automata model, where ENC agents follow simple rules, which reflect the underlying biology of movement, proliferation and differentiation. By simulating ENC precursors and the associated neurons and axons, two models with different fundamental mechanisms are developed. From local rules, a mesoscale network pattern with lacunae emerges, which can be analyzed quantitatively. Simulation and analysis establishes the parameters that affect the morphology of the resulting network. This investigation into the axon/ENC and ENC/ENC interplay suggests possible explanations for observations in mouse and avian embryos in normal and abnormal ENS development, as well as further experimentation.     

Isolation and directed differentiation of neural crest stem cells derived from human embryonic stem cells

Vertebrate neural crest development depends on pluripotent, migratory precursor cells. Although avian and murine neural crest stem (NCS) cells have been identified, the isolation of human NCS cells has remained elusive. Here we report the derivation of NCS cells from human embryonic stem cells at the neural rosette stage. We show that NCS cells plated at clonal density give rise to multiple neural crest lineages. The human NCS cells can be propagated in vitro and directed toward peripheral nervous system lineages (peripheral neurons, Schwann cells) and mesenchymal lineages (smooth muscle, adipogenic, osteogenic and chondrogenic cells). Transplantation of human NCS cells into the developing chick embryo and adult mouse hosts demonstrates survival, migration and differentiation compatible with neural crest identity. The availability of unlimited numbers of human NCS cells offers new opportunities for studies of neural crest development and for efforts to model and treat neural crest-related disorders.     

Effects of low-intensity pulsed ultrasound on cell viability, proliferation and neural differentiation of induced pluripotent stem cells-derived neural crest stem cells

Low-intensity pulsed ultrasound (LIPUS) acting on induced pluripotent stem cells–derived neural crest stem cells (iPSCs–NCSCs) is considered a promising therapy to improve the efficacy of injured peripheral nerve regeneration. Effects of LIPUS on cell viability, proliferation and neural differentiation of iPSCs–NCSCs were examined respectively in this study. LIPUS at 500 mW cm^?2 enhanced the viability and proliferation of iPSCs–NCSCs after 2 days and, after 4 days, up-regulated gene and protein expressions of NF-M, Tuj1, S100β and GFAP in iPSCs–NCSCs whereas after 7 days expression of only NF-M, S100β and GFAP were up-regulated. LIPUS treatment at an appropriate intensity can, therefore, be an efficient and cost-effective method to enhance cell viability, proliferation and neural differentiation of iPSCs–NCSCs in vitro for peripheral nerve tissue engineering.     

Calcineurin initiates smooth muscle differentiation in neural crest stem cells

The process of vascular smooth muscle cell (vSMC) differentiation is critical to embryonic angiogenesis. However, despite its importance, the vSMC differentiation program remains largely undefined. Murine gene disruption studies have identified several gene products that are necessary for vSMC differentiation, but these methodologies cannot establish whether or not a factor is sufficient to initiate the differentiation program. A gain-of-function system consisting of normal vSMC progenitor cells would serve as a useful complement to whole animal loss-of-function studies. We use such a system here, namely freshly isolated rat neural crest stem cells (NCSCs), to show that activation of the calcineurin signaling pathway is sufficient to drive these cells toward a smooth muscle fate. In addition, we present data suggesting that transforming growth factor (TGF)-beta1, which also causes NCSCs to differentiate into smooth muscle, activates calcineurin signaling in NCSCs, leading to a model in which activation of calcineurin signaling is the mechanism by which TGF-beta1 causes SMC differentiation in these cells.     

Delivery of platelet-derived growth factor as a chemotactic factor for mesenchymal stem cells by bone-mimetic electrospun scaffolds

The recruitment of mesenchymal stem cells (MSCs) is a vital step in the bone healing process, and hence the functionalization of osteogenic biomaterials with chemotactic factors constitutes an important effort in the tissue engineering field. Previously we determined that bone-mimetic electrospun scaffolds composed of polycaprolactone, collagen I and nanohydroxyapatite (PCL/col/HA) supported greater MSC adhesion, proliferation and activation of integrin-related signaling cascades than scaffolds composed of PCL or collagen I alone. In the current study we investigated the capacity of bone-mimetic scaffolds to serve as carriers for delivery of an MSC chemotactic factor. In initial studies, we compared MSC chemotaxis toward a variety of molecules including PDGF-AB, PDGF-BB, BMP2, and a mixture of the chemokines SDF-1α, CXCL16, MIP-1α, MIP-1β, and RANTES. Transwell migration assays indicated that, of these factors, PDGF-BB was the most effective in stimulating MSC migration. We next evaluated the capacity of PCL/col/HA scaffolds, compared with PCL scaffolds, to adsorb and release PDGF-BB. We found that significantly more PDGF- BB was adsorbed to, and subsequently released from, PCL/col/HA scaffolds, with sustained release extending over an 8-week interval. The PDGF-BB released was chemotactically active in transwell migration assays, indicating that bioactivity was not diminished by adsorption to the biomaterial. Complementing these studies, we developed a new type of migration assay in which the PDGF-BB-coated bone-mimetic substrates were placed 1.5 cm away from the cell migration front. These experiments confirmed the ability of PDGF-BB-coated PCL/col/HA scaffolds to induce significant MSC chemotaxis under more stringent conditions than standard types of migration assays. Our collective results substantiate the efficacy of PDGF-BB in stimulating MSC recruitment, and further show that the incorporation of native bone molecules, collagen I and nanoHA, into electrospun scaffolds not only enhances MSC adhesion and proliferation, but also increases the amount of PDGF-BB that can be delivered from scaffolds.     

The effects of embryonic retinal neurons on neural crest cell differentiation into Schwann cells

Amongst the many cell types that differentiate from migratory neural crest cells are the Schwann cells of the peripheral nervous system. While it has been demonstrated that Schwann cells will not fully differentiate unless in contact with neurons, the factors that cause neural crest cells to enter the differentiative pathway that leads to Schwann cells are unknown. In a previous paper (Development 105: 251, 1989), we have demonstrated that a proportion of morphologically undifferentiated neural crest cells express the Schwann cell markers 217c and NGF receptor, and later, as they acquire the bipolar morphology typical of Schwann cells in culture, express S-100 and laminin. In the present study, we have grown axons from embryonic retina on neural crest cultures to see whether this has an effect on the differentiation of neural crest cells into Schwann cells. After 4 to 6 days of co-culture, many more cells had acquired bipolar morphology and S-100 staining than in controls with no retinal explant, and most of these cells were within 200 microns of an axon, though not necessarily in contact with axons. However, the number of cells expressing the earliest Schwann cell markers 217c and NGF receptor was not affected by the presence of axons. We conclude that axons produce a factor, which is probably diffusible, and which makes immature Schwann cells differentiate. The factor does not, however, influence the entry of neural crest cells into the earliest stages of the Schwann cell differentiative pathway.     

Cardiac neural crest cells contribute to the dormant multipotent stem cell in the mammalian heart

Arodent cardiac side population cell fraction formed clonal spheroids in serum-free medium, which expressed nestin, Musashi-1, and multi-drug resistance transporter gene 1, markers of undifferentiated neural precursor cells. These markers were lost following differentiation, and were replaced by the expression of neuron-, glial-, smooth muscle cell-, or cardiomyocyte-specific proteins. Cardiosphere-derived cells transplanted into chick embryos migrated to the truncus arteriosus and cardiac outflow tract and contributed to dorsal root ganglia, spinal nerves, and aortic smooth muscle cells. Lineage studies using double transgenic mice encoding protein 0-Cre/Floxed-EGFP revealed undifferentiated and differentiated neural crest-derived cells in the fetal myocardium. Undifferentiated cells expressed GATA-binding protein 4 and nestin, but not actinin, whereas the differentiated cells were identified as cardiomyocytes. These results suggest that cardiac neural crest-derived cells migrate into the heart, remain there as dormant multipotent stem cells-and under the right conditions-differentiate into cardiomyocytes and typical neural crest-derived cells, including neurons, glia, and smooth muscle.     

Control of neural crest cell behavior and migration: Insights from live imaging

Neural crest cells (NCCs) are a remarkable, dynamic group of cells that travel long distances in the embryo to reach their target sites. They are responsible for the formation of craniofacial bones and cartilage, neurons and glia in the peripheral nervous system and pigment cells. Live imaging of NCCs as they traverse the embryo has been critical to increasing our knowledge of their biology. NCCs exhibit multiple behaviors and communicate with each other and their environment along each step of their journey. Imaging combined with molecular manipulations has led to insights into the mechanisms controlling these behaviors. In this Review, we highlight studies that have used live imaging to provide novel insight into NCC migration and discuss how continued use of such techniques can advance our understanding of NCC biology.Key words: live imaging, neural crest, EMT, Rho GTPase, ephrin, PCP signaling, cadherin, VEGFNeural crest cells (NCCs) are a pluripotent population of cells that migrate from the dorsal neuroepithelium and give rise to multiple cell types including neurons and glia of the peripheral nervous system, pigment cells and craniofacial bone and cartilage.¹ An important hallmark of NCCs is their remarkable ability to migrate over long distances and along specific pathways through the embryo. NCC migration begins with an epithelial to mesenchymal transition (EMT), in which NCCs lose adhesions with their neighbors and segregate from the neuroepithelium.^2,3 Following EMT, NCCs acquire a polarized morphology and initiate directed migration away from the neural tube. While migrating along their pathways to their target tissues, NCCs are guided by extensive communication with one another and by other cues from the extracellular environment. Each of these aspects of NCC migration requires precise regulation of cell motile behaviors, although the mechanisms controlling them are still not well understood. A critical step toward understanding the molecular control of NCC motility is characterization of NCC behaviors as they migrate in their native environment. In the past 15 years, multiple studies have analyzed specific behaviors associated with NCCs along the various stages of their journey and have begun to identify molecules controlling these behaviors. In this review we will focus specifically on these studies that employ live imaging and will highlight the strength of live imaging to reveal mechanisms regulating NCC motility and migration pathways.     

Development of mesenchymal stem cells partially originate from the neural crest

Mesenchymal stem cells (MSCs) are a heterogeneous subset of stromal stem cells isolated from many adult tissues. Previous studies reported that MSCs can differentiate to both mesodermal and neural lineages by a phenomenon referred to as ‘‘dedifferentiation’’ or ‘‘transdifferentiation’’. However, since MSCs have only been defined in vitro, much of their development in vivo is still unknown. Here, we prospectively identified MSCs in the bone marrow from adult transgenic mice encoding neural crest-specific P0-Cre/Floxed-EGFP and Wnt1-Cre/Floxed-EGFP. EGFP-positive MSCs formed spheres that expressed neural crest stem cell genes and differentiated into neurons, glial cells, and myofibroblasts. Interestingly, we observed MSCs both in the GFP⁺ and GFP⁻ fraction and found that there were no significant differences in the in vitro characteristics between these two populations. Our results suggest that MSCs in adult bone marrow have at least two developmental origins, one of which is the neural crest.     

Expression of Schwann cell markers by mammalian neural crest cells in vitro

During embryonic development, neural crest cells differentiate into a wide variety of cell types including Schwann cells of the peripheral nervous system. In order to establish when neural crest cells first start to express a Schwann cell phenotype immunocytochemical techniques were used to examine rat premigratory neural crest cell cultures for the presence of Schwann cell markers. Cultures were fixed for immunocytochemistry after culture periods ranging from 1 to 24 days. Neural crest cells were identified by their morphology and any neural tube cells remaining in the cultures were identified by their epithelial morphology and immunocytochemically. As early as 1 to 2 days in culture, approximately one third of the neural crest cells stained with m217c, a monoclonal antibody that appears to recognize the same antigen as rat neural antigen-1 (RAN-1). A similar proportion of cells were immunoreactive in cultures stained with 192-IgG, a monoclonal antibody that recognizes the rat nerve growth factor receptor. The number of immunoreactive cells increased with time in culture. After 16 days in culture, nests of cells, many of which had a bipolar morphology, were present in the area previously occupied by neural crest cells. The cells in the nests were often associated with neurons and were immunoreactive for m217c, 192-IgG and antibody to S-100 protein and laminin, indicating that the cells were Schwann cells. At all culture periods examined, neural crest cells did not express glial fibrillary acidic protein. These results demonstrate that cultured premigratory neural crest cells express early Schwann cell markers and that some of these cells differentiate into Schwann cells. These observations suggest that some neural crest cells in vivo may be committed to forming Schwann cells and will do so provided that they then proceed to encounter the correct environmental cues during embryonic development.     

Comprehensive characterization of neuroblastoma cell line subtypes reveals bilineage potential similar to neural crest stem cells

Background  

Neuroblastic tumors (NBT) derive from neural crest stem cells (NCSC). Histologically, NBT are composed by neuroblasts and Schwannian cells. In culture, neuroblastic (N-), substrate-adherent (S-) and intermediate phenotype (I-) cell subtypes arise spontaneously.