Inhibition of glial maturation by bone morphogenetic protein 2 in a neural crest-derived cell line

Bone morphogenetic proteins (BMPs) regulate developmental decisions in many neural and nonneural lineages. BMPs influence both CNS neuronal and glial development and promote neuronal differentiation in neural crest derivatives. We investigated the actions of BMP2 on glial differentiation in the peripheral nervous system using NCM1 cells, a neural crest-derived cell line with the properties of peripheral glial precursor cells. BMP2 prevented the acquisition of a mature Schwann cell-like morphology, blocking the expression of mature genes and maintaining expression of several early glial markers. We provide evidence that BMP2 activates the GFAP promoter and define signaling pathways underlying this regulation. Our results demonstrate a novel role for BMPs as inhibitors of glial differentiation in the peripheral nervous system and suggest that BMPs may regulate the developmental timing of glial maturation.     

In vitro and in vivo differentiation of boundary cap neural crest stem cells into mature Schwann cells

Boundary cap cells can generate neurons as well as peripheral glia during embryonic development (Maro, G.S., Vermeren, M., Voiculescu, O., Melton, L., Cohen, J., Charnay, P., Topilko, P., 2004. Neural crest boundary cap cells constitute a source of neuronal and glial cells of the PNS. Nat Neurosci. 7 (9), 930-938), and, recently, the boundary cap was shown to contain multipotent stem cells (Hjerling-Leffler, J., Marmigère, F., Heglind, M., Cederberg, A., Koltzenburg, M., Enerb?ck, S., Ernfors, P., 2005. The boundary cap, a source of neural crest stem cells generating multiple sensory neuron subtypes. Development. 132 (11), 2623-2632). The ability of stem cells to generate mature functional glial phenotypes has not been addressed. In this study, we have explored the competence of boundary neural crest stem cells (bNCSCs) to differentiate into mature functional Schwann cells (SCs) in vitro and in vivo. bNCSCs failed to differentiate into SCs in vitro when cultured in a defined media and in vivo when grafted into adult rat sciatic nerves. However, in the presence of neuregulins, during long-term cultures, the majority of bNCSCs differentiated into SCs. After analysis of the in vivo expression of Sox2, Sox10, S100, GFAP, fibronectin and Krox20 in the glial lineages, we used these markers to characterize differentiation of the bNCSCs. Gliogenesis of bNCSCs proceeded similar to that in vivo by sequentially adopting a SC precursor and immature Schwann cell before maturing into myelinating and non-myelinating SCs. In co-culture with explanted dorsal root ganglia (DRG) as well as in vivo in transplants to the axotomized sciatic nerve, these bNCSC-derived SCs myelinated axons as shown by ensheathing of neuronal processes and expression of myelin basic proteins (MBP). These results show that, under appropriate conditions, bNCSCs can generate mature SCs that are functional and can myelinate axons in regenerating nerves.     

Characterization of the trunk neural crest in the bamboo shark,Chiloscyllium punctatum

The neural crest is a population of mesenchymal cells that after migrating from the neural tube gives rise to structure and cell types: the jaw, part of the peripheral ganglia, and melanocytes. Although much is known about neural crest development in jawed vertebrates, a clear picture of trunk neural crest development for elasmobranchs is yet to be developed. Here we present a detailed study of trunk neural crest development in the bamboo shark, Chiloscyllium punctatum. Vital labeling with dioctadecyl tetramethylindocarbocyanine perchlorate (DiI) and in situ hybridization using cloned Sox8 and Sox9 probes demonstrated that trunk neural crest cells follow a pattern similar to the migratory paths already described in zebrafish and amphibians. We found shark trunk neural crest along the rostral side of the somites, the ventromedial pathway, the branchial arches, the gut, the sensory ganglia, and the nerves. Interestingly, C. punctatum Sox8 and Sox9 sequences aligned with vertebrate SoxE genes, but appeared to be more ancient than the corresponding vertebrate paralogs. The expression of these two SoxE genes in trunk neural crest cells, especially Sox9, matched the Sox10 migratory patterns observed in teleosts. Also of interest, we observed DiI cells and Sox9 labeling along the lateral line, suggesting that in C. punctatum, glial cells in the lateral line are likely of neural crest origin. Although this has been observed in other vertebrates, we are the first to show that the pattern is present in cartilaginous fishes. These findings demonstrate that trunk neural crest cell development in C. punctatum follows the same highly conserved migratory pattern observed in jawed vertebrates. J. Comp. Neurol. 521:3303–3320, 2013. © 2013 Wiley Periodicals, Inc.     

Glial fibrillary acidic protein (GFAP) immunoreactivity in enteric ganglia of the chick embryo

We examined by immunohistochemistry the expression of glial fibrillary acidic protein (GFAP) in enteric ganglia of the chick embryo, using a polyclonal antibody. The morphology of enteric ganglion cells was examined by electron microscopy. Faint GFAP immunoreactivity was detected in ganglion cells and cell processes from around day 7 in ovo. Later in development the intensity of the immunofluorescence increased and it became more evident that immunoreactive small ganglion cells (interpreted as primitive glial cells), and their processes, surrounded larger negative cell profiles (interpreted as primitive neuronal cells); GFAP immunofluorescence was also evident in intramuscular and mucosal nerve trunks. In colocalization experiments, GFAP immunoreactivity was detected in a proportion of HNK-1/N-CAM immunoreactive ganglion cells, in both the myenteric and submucosal plexus. In addition, we observed GFAP immunoreactive nerves in wholemount preparations of chick gut from as early as day 4.5 in ovo. In the ganglionated nerve of Remak, GFAP immunoreactive satellite and Schwann cells were in evidence from day 5 of incubation. Neuronal markers, such as neurofilament, have been detected very early in development in neural crest cell populations in chick enteric ganglia. In contrast, the expression of markers of the glial phenotype has previously been observed only in the late stages of embryonic development. From our experiments, we conclude that neuronal and glial phenotypes are immunohistochemically distinct from as early as day 4.5 of incubation, even if by ultrastructural criteria glial cells are clearly distinguishable from neurons only after day 16 in ovo.     

Enteric glia.

The structure of the enteric nervous system (ENS) is different from that of extraenteric peripheral nerve. Collagen is excluded from the enteric plexuses and support for neuronal elements is provided by astrocyte-like enteric glial cells. Enteric glia differ from Schwann cells in that they do not form basal laminae and they ensheath axons, not individually, but in groups. Although enteric glia are rich in the S-100 and glial fibrillary acidic proteins, it has been difficult to find a single chemical marker that distinguishes enteric glia from non-myelinating Schwann cells. Nevertheless, two monoclonal antibodies have been obtained that recognize antigens that are expressed on Schwann cells (Ran-1 in rats and SMP in avians) but not enteric glia. Functional differences between enteric glia and non-myelinating Schwann cells, including responses to gliotoxins and in vitro proliferative rates, have also been observed. Developmentally, enteric glia, like Schwann cells, are derived from the neural crest. In both mammals and birds the precursors of the ENS appear to migrate to the bowel from sacral as well as vagal levels of the crest. These crest-derived emigrés give rise to both enteric glia and neurons; however, analyses of the ontogeny of the enteric innervation in a mutant mouse (the ls/ls), in which the original colonizing waves of crest-derived precursor cells are unable to invade the terminal colon, suggest that enteric glia can also arise from Schwann cells that enter the gut with the extrinsic innervation. When induced to leave back-transplanted segments of avian bowel, enteric crest-derived cells migrate into peripheral nerves and form Schwann cells. Enteric glia and Schwann cells thus appear to be different cell types, but ones that derive from lineages that diverge relatively late in ontogeny.     

Neural crest derived stem cells from dental pulp and tooth-associated stem cells for peripheral nerve regeneration

The peripheral nerve injuries, representing some of the most common types of traumatic lesions affecting the nervous system, are highly invalidating for the patients besides being a huge social burden. Although peripheral nervous system owns a higher regenerative capacity than does central nervous system, mostly depending on Schwann cells intervention in injury repair, several factors determine the extent of functional outcome after healing. Based on the injury type, different therapeutic approaches have been investigated so far. Nerve grafting and Schwann cell transplantation have represented the gold standard treatment for peripheral nerve injuries, however these approaches own limitations, such as scarce donor nerve availability and donor site morbidity. Cell based therapies might provide a suitable tool for peripheral nerve regeneration, in fact, the ability of different stem cell types to differentiate towards Schwann cells in combination with the use of different scaffolds have been widely investigated in animal models of peripheral nerve injuries in the last decade. Dental pulp is a promising cell source for regenerative medicine, because of the ease of isolation procedures, stem cell proliferation and multipotency abilities, which are due to the embryological origin from neural crest. In this article we review the literature concerning the application of tooth derived stem cell populations combined with different conduits to peripheral nerve injuries animal models, highlighting their regenerative contribution exerted through either glial differentiation and neuroprotective/neurotrophic effects on the host tissue.     

A cell surface determinant expressed early on migrating avian neural crest cells

We have produced monoclonal antibodies against quail ciliary ganglion in an attempt to identify specific markers of this neural crest derivative. One of these antibodies, NC/1, recognizes supportive and neuronal cells of the peripheral nervous system and also most, if not all migrating neural crest cells. We report herein the use of NC/1 to identify crest cells during their migration to their site of final localization. In addition, this antibody may shed light on how the neural crest derived mesectoderm and the peripheral nervous system segregate from one another since the NC/1-defined antigen becomes restricted to the cells of the latter.     

A second source of precursor cells for the developing enteric nervous system and interstitial cells of Cajal

The enteric nervous system is believed to be derived solely from the neural crest cells. This is partly based on the belief that the neural crest cells are the sole neural tube-derived cells colonizing the gastrointestinal tract. However, recent studies have shown that after the emigration of neural crest cells an additional population of cells emigrates from the cranial neural tube. These cells originate in the ventral part of the hindbrain, emigrate through the site of attachment of the cranial nerves, and colonize a variety of developing structures including the gastrointestinal tract. This cell population has been named the ventrally emigrating neural tube (VENT) cells. We followed the fate of these cells in the gastrointestinal tract. Ventral hindbrain neural tube cells of chick embryos were tagged with replication-deficient retroviral vectors containing the LacZ gene, after the emigration of neural crest from this region. In control embryos, the viral concentrate was dropped on the dorsal part of the neural tube. Embryos were sacrificed from embryonic days 3–12 and processed for the detection of LacZ positive ventrally emigrating neural tube cells. These cells colonized only the foregut, specifically the duodenum and stomach. Immunostaining with the neural crest cell marker HNK-1 showed that they were HNK-1 negative, indicating that they were not derived from neural crest. Cells were detected in three locations: (1) the myenteric and submucosal plexus of the enteric nervous system; (2) circular smooth muscle cell layer; and (3) mucosal lining of the lumen. A variety of specific markers were used to identify their fate. Some ventrally emigrating neural tube cells differentiated into neurons and glial cells, indicating that the enteric nervous system in the foregut develops from an additional source of precursor cells. It was also found that some of these cells differentiated into interstitial cells of Cajal, which mediate impulses between the enteric nervous system and smooth muscle cells, whereas others differentiated into epithelium. Altogether, these results indicate that the ventrally emigrating neural tube cells are multipotential. More importantly, they reveal a novel source of precursor cells for the neurons and glial cells of the enteric nervous system. The developmental and functional significance of the heterogeneous origin of the cell types remains to be established.     

Gene transfer of lacZ into avian neural tube and neural crest cells by retroviral infection of grafted embryonic tissues

We describe here a new method for transferring genes into cells of the neural tube and neural crest of early avian embryos in vivo. Using the marker gene lacZ as an example, we infected dissected neural tubes from Hamburger-Hamilton stage 12–13 quail embryos with a replication-defective retrovirus carrying lacZ during a 2 hr period of exposure to the virus in culture. Infected neural tubes were then grafted into uninfected host chicken embryos in ovo and, after continued development for several days, the chimeric embryos were processed for β-galactosidase histochemistry to identify the progeny of infected cells. We show that virus-infected neural tubes grafted isotopically into the trunk region of host embryos gave rise to cells of both the spinal cord and neural crest. Infected neural crest cells localized within dorsal root ganglia, sympathetic ganglia, peripheral nerves, and within the skin, where they were likely to give rise to melanocytes. These data are consistent with those using other cell marking techniques applied to the neural crest, indicating that retrovirus infection in culture, grafting, and β-galactosidase expression has a neutral effect on neural crest cell migration and localization. These results indicate the heterospecific grafting of early avian tissues infected with retroviruses carrying foreign genes may be an effective strategy for testing the biological role of various gene products during development. © 1993 Wiley-Liss, Inc.     

Distribution of carbonic anhydrase activity in neurons of the rat

Certain neurons of dorsal root ganglia (DRG) and some fibers of the sciatic nerve contain histochemically demonstrable carbonic anhydrase activity. Since the distribution of this enzyme throughout the nervous system has not yet been evaluated systematically, we conducted a comprehensive histochemical survey focusing particularly on structures derived from the neural crest and nonneural crest ectoderm. In the peripheral nervous system, we observed carbonic anhydrase activity in some, but not all, neurons of dorsal root, trigeminal, celiac, and myenteric ganglia as well as in glial cells throughout the CNS. Some neurons of the nodose ganglion also showed carbonic anhydrase activity. In all first order sensory ganglia that were studied, the enzyme was found only in large (50 micron or above) and medium (20-50 micron) size neurons; in the case of spinal ganglia, the reactive neurons constituted approximately 30% of the total neuronal population. Of these reactive neurons, 56% were heavily stained and 44% were moderately stained. Several possible roles for neuronal carbonic anhydrase are considered.     

Nestin expression defines both glial and neuronal progenitors in postnatal sympathetic ganglia

Sympathetic ganglia are primarily composed of noradrenergic neurons and satellite glial cells. Although both cell types originate from neural crest cells, the identities of the progenitor populations at intermediate stages of the differentiation process remain to be established. Here we report on the identification in vivo of glial and neuronal progenitor cells in postnatal sympathetic ganglia, by using mouse superior cervical ganglia as a model system. There are significant levels of cellular proliferation in mouse superior cervical ganglia during the first 18 days after birth. A majority of the proliferating cells express both nestin and brain lipid-binding protein (BLBP). Bromodeoxyuridine (BrdU) fate-tracing experiments demonstrate that these nestin and BLBP double-positive cells represent a population of glial progenitors for sympathetic satellite cells. The glial differentiation process is characterized by a marked downregulation of nestin and upregulation of S100, with no significant changes in the levels of BLBP expression. We also identify a small number of proliferating cells that express nestin and tyrosine hydroxylase, a key enzyme of catecholamine biosynthesis that defines sympathetic noradrenergic neurons. Together, these results establish nestin as a common marker for sympathetic neuronal and glial progenitor cells and delineate the cellular basis for the generation and maturation of sympathetic satellite cells.     

Central nervous system stem/progenitor cells form neurons and peripheral glia after transplantation to the dorsal root ganglion

We asked whether neural stem/progenitor cells from the cerebral cortex of E14.5 enhanced green fluorescent protein transgenic mice are able to survive grafting and differentiate in the adult rat dorsal root ganglion. Neurospheres were placed in lumbar dorsal root ganglion cavities after removal of the dorsal root ganglia. Alternatively, dissociated neurospheres were injected into intact dorsal root ganglia. Enhanced green fluorescent protein-positive cells in the dorsal root ganglion cavity were located in clusters and expressed beta-III-tubulin or glial fibrillary acidic protein after 1 month, whereas after 3 months, surviving grafted cells expressed only glial fibrillary acidic protein. In the intact adult DRG, transplanted neural stem/progenitor cells surrounded dorsal root ganglion cells and fibers, and expressed glial but not neuronal markers. These findings show that central nervous system stem/progenitor cells can survive and differentiate into neurons and peripheral glia after xenotransplantation to the adult dorsal root ganglion.     

Adult dorsal root ganglia sensory neurons express the early neuronal fate marker doublecortin

It has been widely accepted that doublecortin (DCX) may represent a neuronal fate marker transiently expressed by immature neurons during development of the central and peripheral nervous tissue and in neurogenic areas of the adult brain. Previous work described the presence of DCX in the developing dorsal root ganglia (DRG), structures of the peripheral nervous system originating from the neural crest, but no information is available on its expression in adulthood. To this purpose, we have performed an immunohistochemical and biochemical analysis for DCX expression in DRG from adult male mice and rats. To our surprise, we demonstrated that the majority of DRG neurons do express DCX, both in somata and in fibers. DCX(+) cells have been characterized morphologically and phenotypically with well-established markers of DRG neuronal subpopulations. A large number of DCX(+) cells belong to the small and medium-sized nociceptive neurons. Additionally, DCX immunoreactivity is present in the spinal cord dorsal horns, the projection area of DRG neurons. The novel and unexpected localization for DCX protein opens up new, interesting vistas on the functional role of this protein in mature neurons and in particular in sensory neurons.     

Relationship between differentiation and terminal mitosis: chick sensory and ciliary neurons differentiate after terminal mitosis of precursor cells, whereas sympathetic neurons continue to divide after differentiation

A population of undifferentiated cells has been characterized during the early development of nodose and ciliary ganglia. This population is defined by the absence of surface markers specific for neurons (tetanus toxin receptor, Q211 antigen) and for glial cells (O4 antigen). These undifferentiated cell populations were isolated from the ganglia and were shown to contain neuronal precursor cells that were able to differentiate in vitro into neurons, as characterized by morphology and surface antigens. Undifferentiated cells were detected during the period of neuronal birth, indicating that dividing neuronal precursor cells do not express neuron-specific surface markers. This was directly shown by 3H-thymidine-labeling studies using nodose ganglia, ciliary ganglia, and dorsal root ganglia. In sympathetic ganglia, however, no undifferentiated neuronal precursor cells were detectable at developmental stages when sympathetic neurons are born. 3H-Thymidine injected during that stage at E7 was incorporated into cells expressing the neuronal markers tetanus toxin receptor and Q211 antigen. Quantitative fluorimetric determination of the DNA content of dissociated sympathetic ganglion cells demonstrated the presence of a population of Q211-positive sympathetic ganglion cells in the G2 phase of the cell cycle. E7 sympathetic ganglion cells expressing neuronal surface markers were also shown to be able to divide in vitro. We have concluded that the relationship between terminal mitosis and the onset of differentiation differs between ganglia of the chick peripheral nervous system: Sympathetic ganglion cells continue to divide after the acquisition of neuronal properties, whereas neuronal precursor cells from other autonomic and sensory ganglia start to differentiate after a terminal mitosis.     

Differentiation of radial glia-like cells from embryonic stem cells

Radial glial cells play important roles in neural development. They provide support and guidance for neuronal migration and give rise to neurons and glia. In vitro, neurons, astrocytes, and oligodendrocytes can be generated from neural and embryonic stem cells, but the generation of radial glial cells from these stem cells has not yet been reported. Since the differentiation of radial glial cells is indispensable during brain development, we hypothesize that stem cells also generate radial glial cells during in vitro neural differentiation. To test this hypothesis, we utilized five different clones of mouse embryonic (ES) and embryonal carcinoma (EC) stem cell lines to investigate the differentiation of radial glial cells during in vitro neural differentiation. Here, we demonstrate that radial glia-like cells can be generated from ES/EC cell lines. These ES/EC cell-derived radial glia-like cells are similar in morphology to radial glial cells in vivo, i.e., they are bipolar with an unbranched long process and a short process. They also express several cytoskeletal markers, such as nestin, RC2, and/or GFAP, that are characteristics of radial glial cells in vivo. The processes of these in vitro generated radial glia-like cells are organized into parallel arrays that resemble the radial glial scaffolds in neocortical development. Since radial glia-like cells were observed in all five clones of ES/EC cells tested, we suggest that the differentiation of radial glial cells may be a common pathway during in vitro neural differentiation of ES cells. This novel in vitro model system should facilitate the investigation of regulation of radial glial cell differentiation and its biological function.     

Migration and differentiation of neural crest and ventral neural tube cells in vitro: implications for in vitro and in vivo studies of the neural crest

During vertebrate development, neural crest cells migrate from the dorsal neural tube and give rise to pigment cells and most peripheral ganglia. To study these complex processes it is helpful to make use of in vitro techniques, but the transient and morphologically ill-defined nature of neural crest cells makes it difficult to isolate a pure population of undifferentiated cells. We have used several established techniques to obtain neural crest-containing cultures from quail embryos and have compared their subsequent differentiation. We confirm earlier reports of neural crest cell differentiation in vitro into pigment cells and catecholamine-containing neurons. However, our results strongly suggest that the 5-HT-containing cells that develop in outgrowths from thoracic neural tube explants are not neural crest cells. Instead, these cells arise from ventral neural tube precursors that normally give rise to a population of serotonergic neurons in the spinal cord and, in vitro, migrate from the neural tube. Therefore, results based on previously accepted operational definitions of neural crest cells may not be valid and should be reexamined. Furthermore, the demonstration that cells from the ventral (non-neural crest) part of the neural tube migrate in vitro suggests that the same phenomenon may occur in vivo. We propose that the embryonic "neural trough," as well as the neural crest, may contribute to the PNS of vertebrates.     

Heterogeneity in migrating neural crest cells revealed by a monoclonal antibody

A monoclonal antibody, GlN1, obtained by immunization with extracts of the 14 d embryonic quail nodose ganglion, is described. GlN1 recognizes an antigenic determinant present in virtually all the satellite cells of the peripheral ganglia, all Schwann cells of the peripheral nerves, and in subpopulations of sensory and autonomic neurons of embryonic and adult quails and chickens. The molecular weight of the antigen(s) revealed by GlN1 in embryonic day 12 quail dorsal root ganglion (DRG) cultures is around 80 kDa. In the neural crest, GlN1 determinant is found as soon as the crest cells leave the neural primordium. Only a proportion (25%) of the migrating neural crest cells carry the antigen. This demonstrates that the neural crest is composed of a heterogeneous population of cells from its early migratory stages. Being selectively distributed on neural crest cells and its derivatives, the GlN1 determinant may be considered as a "differentiation antigen" that will be useful in further studies on cell-line segregation during the ontogeny of the PNS.