Early variations of the disialoganglioside GD3 in chicken embryonic brain support its role in cell migration

In the present study a primary culture system of chicken embryo brain neurons was used in the early period of chicken brain development from day 6 until day 8, which was shown to be a suitable model of neuritogenesis, cell migration and reaggregation. Dissociated chicken optic tectum cells from embryonic stage 31 were cultured on polylysine-coated dishes under serum-free conditions up to 3 days. Freshly dissociated neurons developed short processes, which contacted one another and formed fasciculated bundles. Cell somata migrated along the neurite bundles, similar to migrating neurons in vivo, forming three-dimensional tissue-like clusters. This system was used to study the possible functions of the disialoganglioside GD3 for these neuronal differentiation steps. GD3 represents the predominant ganglioside of embryonic neurons before neuritogenesis in vitro and in vivo. Its biosynthesis is followed during day 6 until day 8 of embryonic brain development. Incubation of dissociated neurons with the monoclonal antibody R-24, recognising the GD3 on the cell surface, led to a total blocking of neurite outgrowth. Accordingly, neither cell migration nor reaggregation could be found. These results indicate that the disialoganglioside GD3 plays a central role in neuronal differentiation and development in the embryonic chicken brain.     

Migration Behavior of Granule Cell Neurons in Cerebellar Cultures. II. An Electron Microscopic Study

We examined the fine structure of migrating granule cell neurons in cerebellar microexplant cultures. Radially migrating bipolar cells extended microspikes or small filopodia from their soma and processes and frequently made contact with neighboring cells. These microspikes contained microfilaments but no microtubules. At the later phase of the migration, in which they had symmetrical bipolar long processes, filopodia extending from perikarial region of cells contained microtubules, suggesting that they are precursors of the future thick perpendicular processes. When cell bodies changed orientation from radial to perpendicular, microtubules that were nucleated from perinuclear centrioles frequently extended into both thick radial and perpendicular processes from the perikarial region. Bundles of 10nm intermediate filaments also appeared in these processes. During migration by the perpendicular contact guidance, many filopodia extending from both the thick leading processes and thin trailing processes made close contacts with the radial parallel neurite. These findings suggest that; 1) The direct contact of the filopodia from both the growth cones and their processes of the granule cells to the neurite bundle plays roles in both the parallel and perpendicular contact guidances. 2) The spacial and temporal changes of cytoskeletons and the association of microtubules with perinuclear centrioles are important for the formation of perpendicular processes and initiation of the perpendicular contact guidance.     

Endocytosis regulates cell soma translocation and the distribution of adhesion proteins in migrating neurons

Newborn neurons migrate from their birthplace to their final location to form a properly functioning nervous system. During these movements, young neurons must attach and subsequently detach from their substrate to facilitate migration, but little is known about the mechanisms cells use to release their attachments. We show that the machinery for clathrin-mediated endocytosis is positioned to regulate the distribution of adhesion proteins in a subcellular region just proximal to the neuronal cell body. Inhibiting clathrin or dynamin function impedes the movement of migrating neurons both in vitro and in vivo. Inhibiting dynamin function in vitro shifts the distribution of adhesion proteins to the rear of the cell. These results suggest that endocytosis may play a critical role in regulating substrate detachment to enable cell body translocation in migrating neurons.     

Common precursors for neural and mesectodermal derivatives in the cephalic neural crest.

The cephalic neural crest (NC) of vertebrate embryos yields a variety of cell types belonging to the neuronal, glial, melanocytic and mesectodermal lineages. Using clonal cultures of quail migrating cephalic NC cells, we demonstrated that neurons and glial cells of the peripheral nervous system can originate from the same progenitors as cartilage, one of the mesectodermal derivatives of the NC. Moreover, we obtained evidence that the migrating cephalic NC contains a few highly multipotent precursors that are common to neurons, glia, cartilage and pigment cells and which we interprete as representative of a stem cell population. In contrast, other NC cells, although provided with identical culture conditions, give rise to clones composed of only one or some of these cell types. These cells thus appear restricted in their developmental potentialities compared to multipotent cells. It is therefore proposed that, in vivo, the active proliferation of pluripotent NC cells during the migration process generates distinct subpopulations of cells that become progressively committed to different developmental fates.     

Reaggregation of fetal rat brain cells in a stationary culture system I: Methodology and cell identification

Summary A stationary tissue culture system for reaggregation cultures of rat brain cells is described. Aggregates were formed by placing cells at high concentrations in liquid overlay cultures on a nonadherent nutrient agar surface. No physical stress in the form of rotation or shaking was applied to the aggregating cell population. Transmission electron microscopy and immunohistochemistry showed that the cells developed from homogeneously dispersed, immature cells in Day 4 aggregates, to mature astrocytes, oligodendrocytes, and neurons in Day 20 aggregates. Twenty days and older aggregates had a tightly packed neuropil which was most prominent in a cell-sparse outer layer of the aggregates. When the aggregates were allowed to adhere to a substrate, both glial fibrillary acidic protein (GFAP) positive and negative cells were observed migrating out from the aggregates. Cells giving a positive reaction for neuron specific enolase (NSE) were also present. This reaggregation procedure, with transfer of selected brain cell aggregates into agar-coated multiwells is an alternative three-dimensional culture system which can be potentially useful in the study of morphogenesis and cell interactions in the nervous system. This project was supported by the Norwegian Cancer Society.     

The migratory behavior of immature enteric neurons

While they are migrating caudally along the developing gut, around 10%-20% of enteric neural crest-derived cells start to express pan-neuronal markers and tyrosine hydroxylase (TH). We used explants of gut from embryonic TH-green fluorescence protein (GFP) mice and time-lapse microscopy to examine whether these immature enteric neurons migrate and their mode of migration. In the gut of E10.5 and E11.5 TH-GFP mice, around 50% of immature enteric neurons (GFP(+) cells) migrated, with an average speed of around 15 mum/h. This is slower than the speed at which the population of enteric neural crest-derived cells advances along the developing gut, and hence neuronal differentiation seems to slow, but not necessarily halt, the caudal migration of enteric neural crest cells. Most migrating immature enteric neurons migrated caudally by extending a long-leading process followed by translocation of the cell body. This mode of migration is different from that of non-neuronal enteric neural crest-derived cells and neural crest cells in other locations, but resembles that of migrating neurons in many regions of the developing central nervous system (CNS). In migrating immature enteric neurons, a swelling often preceded the movement of the nucleus in the direction of the leading process. However, the centrosomal marker, pericentrin, was not localized to either the leading process or swelling. This seems to be the first detailed report of neuronal migration in the developing mammalian peripheral nervous system.     

Dynamics of neural crest-derived cell migration in the embryonic mouse gut

Neural crest-derived cells that form the enteric nervous system undergo an extensive migration from the caudal hindbrain to colonize the entire gastrointestinal tract. Mice in which the expression of GFP is under the control of the Ret promoter were used to visualize neural crest-derived cell migration in the embryonic mouse gut in organ culture. Time-lapse imaging revealed that GFP(+) crest-derived cells formed chains that displayed complicated patterns of migration, with sudden and frequent changes in migratory speed and trajectories. Some of the leading cells and their processes formed a scaffold along which later cells migrated. To examine the effect of population size on migratory behavior, a small number of the most caudal GFP(+) cells were isolated from the remainder of the population. The isolated cells migrated slower than cells in large control populations, suggesting that migratory behavior is influenced by cell number and cell-cell contact. Previous studies have shown that neurons differentiate among the migrating cell population, but it is unclear whether they migrate. The phenotype of migrating cells was examined. Migrating cells expressed the neural crest cell marker, Sox10, but not neuronal markers, indicating that the majority of migratory cells observed did not have a neuronal phenotype.     

A novel approach to selectively target neuronal subpopulations reveals genetic pathways that regulate tangential migration in the vertebrate hindbrain

Vertebrate genes often play functionally distinct roles in different subsets of cells; however, tools to study the cell-specific function of gene products are poorly developed. Therefore, we have established a novel mouse model that enables the visualization and manipulation of defined subpopulations of neurons. To demonstrate the power of our system, we dissected genetic cascades in which Pax6 is central to control tangentially migrating neurons of the mouse brainstem. Several Pax6 downstream genes were identified and their function was analyzed by over-expression and knock-down experiments. One of these, Pou4f2, induces a prolonged midline arrest of growth cones to influence the proportion of ipsilaterally versus contralaterally settling neurons. These results demonstrate that our approach serves as a versatile tool to study the function of genes involved in cell migration, axonal pathfinding, and patterning processes. Our model will also serve as a general tool to specifically over-express any gene in a defined subpopulation of neurons and should easily be adapted to a wide range of applications.     

Migration of neurons between ganglia in the metamorphosing insect nervous system

Migration of neurons over long distances occurs during the development of the adult central nervous system of the sphinx moth Manduca sexta, and the turnip moth Agrotis segetum. From each of the suboesophageal and three thoracic ganglia, bilaterally-paired clusters of immature neurons and associated glial cells migrate posteriorly along the interganglionic connectives, to enter the next posterior ganglion. The first sign of migration is observed at the onset of metamorphosis, when posterio-lateral cell clusters gradually separate from the cortex of neuronal cell bodies and enter the connectives. Cell clusters migrate posteriorly along the connective to reach the next ganglion over the first three days (approximately 15%) of pupal development. During migration, each cell cluster is completely enveloped by a single giant glial cell spanning the entire length of the connective between two adjacent ganglia. Intracellular cobalt staining reveals that each migrating neuron has an ovoid cell body and an extremely long leading process which extends as far as the next posterior ganglion; this is not a common morphology for migrating neurons that have been described in vertebrates. Once the cells arrive at the anterior cortex of the next ganglion, they rapidly intermingle with the surrounding neurons and so we were unable to determine the fate of the migrating neurons at their final location.     

Doublecortin is a developmentally regulated, microtubule-associated protein expressed in migrating and differentiating neurons.

Recently, we and others reported that the doublecortin gene is responsible for X-linked lissencephaly and subcortical laminar heterotopia. Here, we show that Doublecortin is expressed in the brain throughout the period of corticogenesis in migrating and differentiating neurons. Immunohistochemical studies show its localization in the soma and leading processes of tangentially migrating neurons, and a strong axonal labeling is observed in differentiating neurons. In cultured neurons, Doublecortin expression is highest in the distal parts of developing processes. We demonstrate by sedimentation and microscopy studies that Doublecortin is associated with microtubules (MTs) and postulate that it is a novel MAP. Our data suggest that the cortical dysgeneses associated with the loss of Doublecortin function might result from abnormal cytoskeletal dynamics in neuronal cell development.     

Netrin 1 acts as an attractive or as a repulsive cue for distinct migrating neurons during the development of the cerebellar system

Netrin 1 is a long-range diffusible factor that exerts chemoattractive or chemorepulsive effects on developing axons growing to or away from the neural midline. Here we used tissue explants to study the action of netrin 1 in the migration of several cerebellar and precerebellar cell progenitors. We show that netrin 1 exerts a strong chemoattractive effect on migrating neurons from the embryonic lower rhombic lip at E12-E14, which give rise to precerebellar nuclei. Netrin 1 promotes the exit of postmitotic migrating neurons from the embryonic lower rhombic lip and upregulates the expression of TAG-1 in these neurons. In addition, in the presence of netrin 1, the migrating neurons are not isolated but are associated with thick fascicles of neurites, typical of the neurophilic way of migration. In contrast, the embryonic upper rhombic lip, which contains tangentially migrating granule cell progenitors, did not respond to netrin 1. Finally, in the postnatal cerebellum, netrin 1 repels both the parallel fibres and migrating granule cells growing out from explants taken from the external germinal layer. The developmental patterns of expression in vivo of netrin 1 and its receptors are consistent with the notion that netrin 1 secreted in the midline acts as chemoattractive cue for precerebellar neurons migrating circumferentially along the extramural stream. Similarly, the pattern of expression in the postnatal cerebellum suggests that netrin 1 could regulate the tangential migration of postmitotic premigratory granule cells. Thus, molecular mechanisms considered as primarily involved in axonal guidance appear also to steer neuronal cell migration.     

Intercellular adhesion can be visualized using fluorescently labeled fibrosarcoma HT1080 cells cocultured with hematopoietic cell lines or CD34(+) enriched human mobilized peripheral blood cells

BACKGROUND: Intercellular contacts between adjacent cells migrating over each other are important in many cellular processes. However, it has been difficult to visualize and identify dynamic intercellular adhesions between migrating cells in situ. METHODS: Two fluorescent membrane dyes, PKH2 and PKH26 for staining HT1080 and hematopoietic cells and cell lines, and an automated fluorescence microscopy system were used to monitor intercellular adhesion. RESULTS: Cellular extensions connecting two or more adjacent cells were visualized, showing the intercellular adhesion between migrating cells for minutes and up to hours. After cells adhered to each other, followed by cell migration in different directions, cellular extensions were dragged from the pivotal contact points in different focal planes. CD34(+)-enriched mobilized peripheral blood cells and six hematopoietic cell lines showed intercellular connections in cocultures with HT1080. However, the frequency of intercellular connections was variable in different cocultures. A cell density of about 3.1 x 10(4) cells/cm(2) for both cell lines in cocultures provided an adequate number of cells in each field of view, showing up to four intercellular connections per 100 total cells plated. DISCUSSION: The tools derived from this study will open new areas of investigation for understanding the mechanism of the intercellular adhesion process.     

Biochemical and functional characterization of a novel neuron-glia adhesion molecule that is involved in neuronal migration

Adhesion molecule on glia (AMOG) is a novel neural cell adhesion molecule that mediates neuron-astrocyte interaction in vitro. In situ AMOG is expressed in the cerebellum by glial cells at the critical developmental stages of granule neuron migration. Granule neuron migration that is guided by surface contacts between migrating neurons and astroglial processes is inhibited by monoclonal AMOG antibody, probably by disturbing neuron-glia adhesion. AMOG is an integral cell surface glycoprotein of 45-50-kD molecular weight with a carbohydrate content of at least 30%. It does not belong to the L2/HNK-1 family of neural cell adhesion molecules but expresses another carbohydrate epitope that is shared with the adhesion molecules L1 and myelin-associated glycoprotein, but is not present on N-CAM or J1.     

Isolation of radial glial cells by fluorescent-activated cell sorting reveals a neuronal lineage

The developing central nervous system of vertebrates contains an abundant cell type designated radial glial cells. These cells are known as guiding cables for migrating neurons, while their role as precursor cells is less clear. Since radial glial cells express a variety of astroglial characteristics and differentiate as astrocytes after completing their guidance function, they have been considered as part of the glial lineage. Using fluorescence-activated cell sorting, we show here that radial glial cells also are neuronal precursors and only later, after neurogenesis, do they shift towards an exclusive generation of astrocytes. These results thus demonstrate a novel function for radial glial cells, namely their ability to generate two major cell types found in the nervous system, neurons and astrocytes.     

Development and persistence of catecholaminergic neurons in cultured explants of fetal murine vagus nerves and bowel

Transient catecholaminergic (TC) cells have been found to appear in the vagal pathway and bowel of fetal mice and rats. It has been proposed that these cells are migrating vagal crest-derived precursors of enteric neurons that lose their catecholaminergic properties when they terminally differentiate. In the current experiments, segments of fetal mouse gut were explanted before (day E9) TC cells or any neural markers could be detected in situ. Tyrosine hydroxylase (TH)-immunoreactive neurons developed in vitro in 4/12 such explants; therefore, cells with a catecholaminergic potential are present in the gut of at least some animals prior to the in situ expression of this phenotype. The neurogenic potential of cells in the vagal pathway was similarly tested by studying cultures of explanted vagus nerves (day E11). These studies revealed that neural precursors were present in the vagi and gave rise in vitro to neurons that displayed acetylcholinesterase (AChE) activity and neuron-specific enolase (NSE) immunoreactivity. A subset of these neural precursors were capable of migrating and formed satellite ganglia at a distance from the explants. Coincident expression of NSE and TH immunoreactivities was observed, indicating that at least some of the neurons that developed in vitro were derived from TC cells. Vagal TC cells, therefore, are neurogenic. Catecholaminergic cells did not disappear from cultured explants of vagus nerves or gut provided that these tissues contained TC cells at the time of explantation. Instead, catecholaminergic neurons developed and persisted in vitro for as long as cultures were maintained. These neurons contained aromatic L-amino acid decarboxylase as well as TH, NSE and neurofilament immunoreactivities. In contrast, if the bowel was explanted after the in situ disappearance of TC cells, catecholaminergic cells did not arise in the cultures. These experiments indicate that the period of time during which a catecholaminergic phenotype is expressed by neural precursors in the fetal vagal pathway and gut is not fixed, but can be changed by altering the environment of the cells as occurs when the bowel is grown in vitro; moreover, contact with non-neuronal cells within the bowel is not by itself sufficient to inactivate catecholaminergic expression. The nature of the signal responsible for loss of the catecholaminergic phenotype in situ remains to be determined; however, the persistence of catecholaminergic expression in vitro should facilitate the investigation of this signal.     

Studies on the gastrulation of amphibian embryos: Ultrastructure of the migrating cells of anurans

Summary In order to investigate the ultrastructure of the migrating cells in anuran gastrulae, three anurans, which belong to three different genera, were observed with transmission electron microscopy supported by light microscopy of the 1 m sections and scanning electron microscopy. Fine filopodial cell processes, as well as cell processes probably flattened against the inner surface of the blastocoel wall, were formed by the migrating cells. Blebs and lobopodial cell processes were frequently observed inBufo, sometimes inXenopus, but not observed inRana. Microfilaments were observed in the cell processes. Focal close contacts, probably having adhesive properties, were made between the migrating cells and the inner surface of the blastocoel wall. These observations suggest that the cells migrate along the inner surface of the blastocoel wall by forming filopodia and pseudopodia flattened against the wall. The role of the blebs and lobopodial cell processes requires more investigation.     

Evidence for recent as well as long term activation of T cells migrating through endothelial cell monolayers in vitro.

As T cells actively extravasate from blood, they adhere to endothelium and then migrate out of the vessel with a locomotive activity. Although both adhesion and locomotion are properties associated with activated T cells, the two processes are not necessarily associated with identical activation states. Using human endothelial cells (EC) cultured to confluence on collagen gel, we examined the activation state of human peripheral blood T cells that adhere to and migrate through EC monolayers with three different methods: flow cytometric analysis of cell surface activation-related molecules, incorporation of tritiated nucleotide, and cell cycle analysis. The results were as follows. 1) Although expression of very late activation Ag integrins VLA-2 and VLA-3 by the initial blood T cell population (unseparated cells) and of adherent T cells was minimal, 40 to 45% of migrating cells were positive for VLA-2 and VLA-3. 2) The percentage of IL-2R+ cells in both unseparated and adherent cells was below 5% whereas the percentage of IL-2R+ cells among the migrating cells was 22 +/- 9% (range, 12 to 31%, n = 6). 3) Migrating cells expressed the highest CD26, whereas CD26 of adherent (nonmigrating) cells was divided into negative and high expression; in contrast, leukocyte adhesion molecule-1 (L-selectin) of both adherent and migrating cells was mostly low or negative. 4) [3H]Uridine incorporation of migrating and adherent cells was 2.1- to 2.5-fold and 1.4- to 1.7-fold higher, respectively, than that of unseparated cells, indicating that RNA synthesis of migrating cells as well as adherent cells was enhanced. 5) Cell cycle analysis showed that 23.5% of migrating cells appeared to enter the G1 phase but not S or G2 + M phases whereas 2.2% of unseparated cells and 8.0% of adherent cells that did not migrate had an RNA content consistent with entry into G1. These results suggest that cells migrating from normal human blood through unactivated EC have been activated recently as well as showing evidence of long term activation. The activation state of migrating cells is consistent with the hypothesis that previous in vivo activation is required for cells to migrate through EC in this system.     

Serotonin promotes G(o)-dependent neuronal migration in Caenorhabditis elegans

BACKGROUND: The directed migration of neurons during development requires attractive and repulsive cues that control the direction of migration as well as permissive cues that potentiate cell motility and responsiveness to guidance molecules. RESULTS: Here, we show that the neurotransmitter serotonin functions as a permissive signal for embryonic and postembryonic neuronal migration in the nematode C. elegans. In serotonin-deficient mutants, the migrations of the ALM, BDU, SDQR, and AVM neurons were often foreshortened or misdirected, indicating a serotonin requirement for normal migration. Moreover, exogenous serotonin could restore motility to AVM neurons in serotonin-deficient mutants as well as induce AVM-like migrations in the normally nonmotile neuron PVM; this indicates that serotonin was functioning as a permissive cue to enable neuronal motility. The migration defects of serotonin-deficient mutants were mimicked by ablations of serotonergic neuroendocrine cells, implicating humoral release of serotonin in these processes. Mutants defective in G(q) and G(o) signaling, or in N-type voltage-gated calcium channels, showed migration phenotypes similar to serotonin-deficient mutants, and these molecules appeared to genetically function downstream of serotonin in the control of neuronal migration. CONCLUSIONS: Thus, serotonin is important for promoting directed neuronal migration in the developing C. elegans nervous system. We hypothesize that serotonin may promote cell motility through G protein-dependent modulation of voltage-gated calcium channels in the migrating cell.     

Different isoforms of fasciclin II play distinct roles in the guidance of neuronal migration during insect embryogenesis

During the formation of the enteric nervous system (ENS) of the moth Manduca sexta, identified populations of neurons and glial cells participate in precisely timed waves of migration. The cell adhesion receptor fasciclin II is expressed in the developing ENS and is required for normal migration. Previously, we identified two isoforms of Manduca fasciclin II (MFas II), a glycosyl phosphatidylinositol-linked isoform (GPI-MFas II) and a transmembrane isoform (TM-MFas II). Using RNA and antibody probes, we found that these two isoforms were expressed in cell type-specific patterns: GPI-MFas II was expressed by glial cells and newly generated neurons, while TM-MFas II was confined to differentiating neurons. The expression of each isoform also corresponded to the motile state of the different cell types: GPI-MFas II was detected on tightly adherent or slowly spreading cells, while TM-MFas II was expressed by actively migrating neurons and was localized to their most motile regions. Manipulations of each isoform in embryo culture showed that they played distinct roles: whereas GPI-MFas II acted strictly as an adhesion molecule, TM-MFas II promoted the motility of the EP cells as well as maintaining fasciculation with their pathways. These results indicate that precisely regulated patterns of isoform expression govern the functions of fasciclin II within the developing nervous system.     

Solitary hypothalamic neurons inherently express vasopressin and tyrosine hydroxylase

Hypothalamic neurons were grown as single cells in three-dimensional culture. Solitary neurons lacking cell contacts were immunocytochemically examined for inherent expression of vasopressin (VP), tyrosine hydroxylase (TH), and luteinizing hormone releasing hormone (LHRH). Immunoreactive VP and TH were detected within a day. Sixty to eighty-five percent of neurons displayed homogeneously distributed reaction product for VP or TH. One percent exhibited intense punctate staining of somas and varicosities. Few neurons stained for LHRH. Results indicate that hypothalamic neurons can express appropriate neuropeptides and transmitter-specific products without contacting other neurons or nonneuronal cells. Thus, this culture system may provide a useful model to study intrinsic neuronal processes.