Free radicals and ethanol-induced cytotoxicity in neural crest cells

Associations between ethanol-induced cranial neural crest cell (NCC) damage in mammalian embryos and subsequent malformations as observed in human fetal alcohol syndrome have previously been illustrated. The vulnerability of NCCs to this teratogen may result, at least in part, from their sensitivity to free radical damage. To examine relationships between free radical generation and NCC cytotoxicity, primary culture of mouse NCCs was used. NCC viability was determined in both dose- and time-response studies involving ethanol exposure. After 48 hr of culture, cell viability was significantly diminished at all doses tested (i.e., 50, 100, 150, and 200 mM ethanol). At 100 mM ethanol (a dosage that is teratogenic in vivo and in whole embryo culture), cell viability decreased to approximately 50% of control values over the first 12 hr of culture, and decreased further, to approximately 20% by 48 hr. Using nitroblue tetrazolium as a probe, it was observed that exposure of NCCs to ethanol stimulated the production of superoxide anion radicals. Co-treatment of the ethanol-exposed NCCs with free radical scavengers including 300 units/ml of superoxide dismutase, catalase (500 units/ml), or alpha-tocopherol (300 microM) significantly improved NCC viability. These results suggest that the ethanol-induced NCC injury is mediated, at least in part, through the generation of free radicals. To test this hypothesis further, NCCs were exposed in culture to xanthine/xanthine oxidase. Exogenous free radicals generated by the xanthine/xanthine oxidase system resulted in reduced NCC viability, the severity of which increased in a time and enzyme concentration-related manner. Superoxide dismutase (300 units/ml) and catalase (500 units/ml) significantly reduced the effects of the xanthine/xanthine oxidase-generated free radicals on NCC viability. The similarity between the susceptibility of NCCs to ethanol and their susceptibility to exogenous free radicals in concert with the free radical scavenger-mediated amelioration of ethanol and exogenous free radical-induced NCC death strongly suggest that free radicals play a significant role in ethanol-induced NCC death.     

Retinoic acid induces changes in the rhombencephalic neural crest cells migration and extracellular matrix composition in chick embryos

Studies on haemopoietic stem cells had led to the realisation that negative feedback inhibitors play an important role in regulating their proliferation. One such molecule was identified as MIP-1 alpha. One of a family of cytokines, originally recognised as inflammatory molecules, MIP-1 alpha is now potentially valuable as a means of manipulating and protecting haemopoietic (and possibly other) stem cells during chemotherapy. This short review briefly considers the structural classification of MIP-1 alpha and its molecular relatives and indicates some of the probable human/murine equivalent molecules outlining the evidence for the equivalence of MIP-1 alpha (murine) and LD78 (human). Sources of MIP-1 alpha/LD78 are identified as monocyte/macrophage and lymphocytic cells and their role in inflammatory responses is seen to be significant. All proliferation in haemopoietic tissue is now recognised as a major target for MIP-1 alpha action. In vitro it synergises with certain growth factors to promote progenitor cell colony formation, but effects are dependent on the maturational age of the cells promoted. With more primitive cells it is seen as inhibitory. This property is particularly valuable in vivo where MIP-1 alpha can protect stem cells against the effects of cytotoxic agents. Since it appears that leukaemic stem cell proliferation is not inhibited, MIP-1 alpha/LD78 present great potential for stem cell protection in the theatre of cytotoxic therapies.     

The delayed entry of thoracic neural crest cells into the dorsolateral path is a consequence of the late emigration of melanogenic neural crest cells from the neural tube

Recent studies have shown that changes in dendritic architecture are an important component of functional plasticity in the adult central nervous system. In the present study, we determined whether gonadectomy induces changes in dendritic architecture in the arcuate nucleus, a target tissue for gonadal hormones. A combination of retrograde labeling with systemically injected Fluoro-Gold and intracellular injection of neurons in a fixed-slice preparation was used to examine the morphology of neuroendocrine neurons in the rat arcuate nucleus. Intracellullary filled arcuate neuroendocrine neurons (8-21 neurons per brain) from intact (n = 5) and orchidectomized (n = 5) animals were reconstructed with the aid of a computer microscope. A quantitative analysis revealed that orchidectomy had no effect on the number and distribution of Fluoro-Gold-labeled neuroendocrine neurons in the rat arcuate nucleus. The morphology of arcuate neuroendocrine neurons in intact animals was relatively simple, with the majority of neurons (79%) having only two primary dendrites and few dendritic spines. Compared with intact controls, arcuate neuroendocrine neurons in the orchidectomized group had significantly larger somatic profile areas and exhibited significant increases in dendrite length, dendrite volume, terminal branch number, and spines per unit length of dendrite. The increase in terminal branch number in orchidectomized animals was due primarily to the appearance of short branches that gave a striking, claw-like appearance to many of the distal dendrites. These results provide evidence for hormonal regulation of dendritic morphology of arcuate neuroendocrine neurons in adult mammals.     

A role for rhoB in the delamination of neural crest cells from the dorsal neural tube

The differentiation of neural crest cells from progenitors located in the dorsal neural tube appears to involve three sequential steps: the specification of premigratory neural crest cell fate, the delamination of these cells from the neural epithelium and the migration of neural crest cells in the periphery. BMP signaling has been implicated in the specification of neural crest cell fate but the mechanisms that control the emergence of neural crest cells from the neural tube remain poorly understood. To identify molecules that might function at early steps of neural crest differentiation, we performed a PCR-based screen for genes induced by BMPs in chick neural plate cells. We describe the cloning and characterization of one gene obtained from this screen, rhoB, a member of the rho family GTP-binding proteins. rhoB is expressed in the dorsal neural tube and its expression persists transiently in migrating neural crest cells. BMPs induce the neural expression of rhoB but not the more widely expressed rho family member, rhoA. Inhibition of rho activity by C3 exotoxin prevents the delamination of neural crest cells from neural tube explants but has little effect on the initial specification of premigratory neural crest cell fate or on the later migration of neural crest cells. These results suggest that rhoB has a role in the delamination of neural crest cells from the dorsal neural tube.     

In vitro system for differentiating pluripotent neural crest cells into smooth muscle cells

The change in vascular smooth muscle cells (SMC) from a differentiated to a dedifferentiated state is the critical phenotypic response that promotes occlusive arteriosclerotic disease. Despite its importance, research into molecular mechanisms regulating smooth muscle differentiation has been hindered by the lack of an in vitro cell differentiation system. We identified culture conditions that promote efficient differentiation of Monc-1 pluripotent neural crest cells into SMC. Exclusive Monc-1 to SMC differentiation was indicated by cellular morphology and time-dependent induction of the SMC markers smooth muscle alpha-actin, smooth muscle myosin heavy chain, calponin, SM22alpha, and APEG-1. The activity of the SM22alpha promoter was low in Monc-1 cells. Differentiation of these cells into SMC caused a 20-30-fold increase in the activity of the wild-type SM22alpha promoter and that of a hybrid promoter containing three copies of the CArG element. By gel mobility shift analysis, we identified new DNA-protein complexes in nuclear extracts prepared from differentiated Monc-1 cells. One of the new complexes contained serum response factor. This Monc-1 to SMC model should facilitate the identification of nodal regulators of smooth muscle development and differentiation.     

Regulative interactions in zebrafish neural crest

Zebrafish trunk neural crest cells that migrate at different times have different fates: early-migrating crest cells produce dorsal root ganglion neurons as well as glia and pigment cells, while late-migrating crest cells produce only non-neuronal derivatives. When presumptive early-migrating crest cells were individually transplanted into hosts such that they migrated late, they retained the ability to generate neurons. In contrast, late-migrating crest cells transplanted under the same conditions never generated neurons. These results suggest that, prior to migration, neural crest cells have intrinsic biases in the types of derivatives they will produce. Transplantation of presumptive early-migrating crest cells does not result in production of dorsal root ganglion neurons under all conditions suggesting that these cells require appropriate environmental factors to express these intrinsic biases. When early-migrating crest cells are ablated, late-migrating crest cells gain the ability to produce neurons, even when they migrate on their normal schedule. Interactions among neural crest cells may thus regulate the types of derivatives neural crest cells produce, by establishing or maintaining intrinsic differences between individual cells.     

Age changes in cells in Streptococcus diacetilactis cultures

The activity of metabolism changed in the cultures of Streptococcus diacetilactis, strain Bogdan, with aging. The number of viable cells decreased as well as the ability to evolve oxygen, to produce C-4 compounds, and to react to pH changes by liberating acids upon alkalifying or neutral products upon acidifying. After the culture had been grown during 72 hours, 2,3-butyleneglycol was found in the cultural broth, and the number of viable cells was low. As was revealed by electron microscopy, the cells were intact during 48 hours; they started to disintegrate by 72 hours. After 120 hours all cells were disintegrated. First, the cells produced no more mesosomes, became less electron dense; then the cell wall was decomposed and the contents of the cell poured through disruptions in the cytoplasmic membrane.     

Mechanisms of neural crest cell migration

Neural crest cells are remarkable in their extensive and stereotypic patterns of migration. The pathways of neural crest migration have been documented by cell marking techniques, including interspecific neural tube grafts, immunocytochemistry and DiI-labelling. In the trunk, neural crest cells migrate dorsally under the skin or ventrally through the somites, where they move in a segmental fashion through the rostral half of each sclerotome. The segmental migration of neural crest cells appears to be prescribed by the somites, perhaps by an inhibitory cue from the caudal half. Within the rostral sclerotome, neural crest cells fill the available space except for a region around the notochord, suggesting the notochord may inhibit neural crest cells in its vicinity. In the cranial region, antibody perturbation experiments suggest that multiple cell-matrix interactions are required for proper in vivo migration of neural crest cells. Neural crest cells utilize integrin receptors to bind to a number of extracellular matrix molecules. Substrate selective inhibition of neural crest cell attachment in vitro by integrin antibodies and antisense oligonucleotides has demonstrated that they possess at least three integrins, one being an alpha 1 beta 1 integrin which functions in the absence of divalent cations. Thus, neural crest cells utilize complex sets of interactions which may differ at different axial levels.     

Origins of neural crest cell diversity

The effect of diets containing 50% of fat calories from butter, butter enriched with mono- and polyunsaturated fatty acids, and margarines with and without trans fatty acids on the serum lipids of 38 healthy men in a free-living condition have been determined. Serum lipid responses to the high level of individual dietary fats were unexpectedly small. The butter diet produced a small, but significant rise (5%) in the total serum cholesterol and low density lipoprotein (LDL)-cholesterol, relative to all other diets. Enrichment of butter with either olive oil (50/50) or sunflower oil (50/50) failed to reduce serum lipid levels below habitual diet values. Hard margarine, containing 29% trans fatty acids, caused a decrease in apolipoprotein A-I and B levels, but did not change total serum cholesterol or LDL-cholesterol levels, relative to habitual diet values. A soft margarine, high in linoleate, with no trans fatty acids reduced total cholesterol, LDL-cholesterol, and apolipoprotein B significantly, relative to all diets. Soft margarine high density lipoprotein (HDL)-cholesterol levels remained unchanged, but apolipoprotein A-I values were decreased relative to habitual and butter diets. The quantities of saturated fatty acids and the sum of monounsaturated and polyunsaturated fatty acids consumed on the hard and soft margarines were equal; therefore, the different response of serum cholesterol and LDL-cholesterol between these two diets is attributable to the trans fatty acids in the hard margarine. The data indicate that trans fatty acids are not metabolically equivalent to the natural cis isomers and that they affect the serum lipid profile adversely.     

BMP-2 and BMP-4, but not BMP-6, increase the number of adrenergic cells which develop in quail trunk neural crest cultures

Isolated rat liver mitochondria were incubated under various metabolic conditions to determine their membrane potential (MMP) as measured continuously by a tetraphenylphosphonium (TPP+)-selective electrode. By flow cytometry, a parallel analysis of fluorescence emissions observing single mitochondria stained with the lipophilic cation 5,5',6,6'-tetrachloro-1,1'3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1) revealed linear correlation between the median orange fluorescence (FL2) due to J-aggregate formations and MMP values measured by TPP+. No correlation was detected with the green fluorescence (FL1) emission. A significantly higher correlation appeared between the FL2/FL1 ratio and MMP values. Within the same mitochondrial population, cytofluorimetric analysis revealed the presence of various classes of organelles with different MMP, whose distribution was dependent on metabolic condition. The highest functional heterogeneity was found in deenergized mitochondria, while the highest homogeneity was observed during the first phase of the phosphorylative process. Thus, these data suggest that the cytofluorimetric use of JC-1 provides direct experimental evidence for the hypothesis of functional mitochondrial heterogeneity, at least with respect to their membrane potential.     

Malignant skin tumors of neural crest origin

In a series of 90,000 consecutive surgical biopsies, ten malignant skin tumors were found that revealed at least two of three tissue components known to be derivatives of the neural crest, namely melanotic cells, neuroid structures, and chondrified tissue. The material studied supports a hypothesis presented earlier, that in spite of their morphologic variegation, such tumors from an entity defined by their common origin, the neural crest.     

Cytoskeletal characterization of arteriovenous epithelioid cells

BACKGROUND: Head and neck osteosarcoma is a comparatively rare and aggressive malignancy. Our goal was to examine the experience of head and neck osteosarcoma patients seen over a 15-year period at the University of Washington Medical Center and compare this with the published experience of other centers in terms of demographics, histology, treatment, and survival rate. METHODS: We reviewed surgical pathology slides and clinical treatment records of 13 patients who were treated at the University of Washington Medical Center between 1981 and 1996. A total of 17 cases from 13 patients (13 primary tumors and 4 recurrences) were studied. RESULTS: There was a slight male predominance, with a male:female ratio of 1.6:1, and median age at diagnosis of 40.9 years (range 22 to 75 years), both slightly higher than has been generally reported. Three of 13 patients had recognized risk factors for the development of osteosarcoma: 2 with a history of prior radiotherapy and 1 with Paget's disease. All surgical pathology specimens were examined independently by two pathologists for histologic grading and typing. At initial presentation, 9/13 (69%) cases had conventional (osteoblastic) histology; 2/13 (15%) were fibroblastic, 1 chondroblastic (8%) and 1 parosteal (8%). Eight of 13 (62%) cases were high grade at initial presentation. Four of 13 (30%) of the primary tumors were low grade 2, of which did not recur over a median follow-up period of 24 months. The other 2 low-grade tumors later recurred locally, as high-grade osteosarcomas, after disease-free intervals of 1 year and 14 years, respectively. One patient had an intermediate-grade tumor which has not recurred as of last follow-up. Combined-modality treatment, including surgery with or without radiotherapy and/or chemotherapy, was given depending on the histologic grade, surgical margins, and recurrence. Some patients with low-grade tumors had surgery only. There were 5 local recurrences, 1 of these following a disease-free interval of 14 years. One patient had 3 separate recurrences at the same site. Ten of 13 (77%) are alive and disease-free. Of the 3 deaths, 1 was related to radiation-induced brain necrosis, without evidence of recurrent tumor. The project 5-year overall survival in this series is 72%, with a mean follow-up of 58 months (median, 36 months). Of those receiving neoadjuvant chemotherapy, 6/7 have survived to the present. CONCLUSION: Given the limitations of a small patient population, our data suggest that neoadjuvant chemotherapy may provide benefit in terms of survival. Longer follow-up will be necessary to support this conclusion. Our data also show that our population has a higher-than-average age of onset, low presence of risk factors, and better survival rate in comparison with the published series from other institutions.     

Effects of mitomycin C in postnatal tooth development in mice with special reference to neural crest cells

Neural crest cells in the pre- and postnatal development of mice showed a specific sensitivity to mitomycin C. In mice injected with mitomycin C 24-48 h after birth and surviving up to the 9th day, a free or attached denticle in the pulp and a fibroma or excessive cell proliferation in the dental follicle or the periodontal ligament were seen; further, a dentinoma developed.     

Analysis of neural crest cell migration in Splotch mice using a neural crest-specific LacZ reporter

In the last decades, several therapies for unipolar depression have been developed, for example cognitive therapy, behavior therapy and pharmacotherapy. A new kind of therapy is bibliotherapy. What is new in this treatment modality is not the content, because bibliotherapy usually uses a cognitive-behavioral approach. Only the form in which it is presented is new. In bibliotherapy the patient takes a standardized treatment home, in book form, and works it through more or less independently. Contacts with therapists are only supportive or facilitative. No traditional relationship between therapist and patient is developed. In this article the relevance of bibliotherapy for the clinical practice is presented and a meta-analysis of the research into bibliotherapy is described.     

Developing rat Purkinje cells are more vulnerable to alcohol-induced depletion during differentiation than during neurogenesis

This study compared the extent of cerebellar Purkinje cell depletion induced by administering alcohol to rats during two temporally distinct periods of Purkinje cell development--neurogenesis and early differentiation. One group received alcohol (5 g/kg/day) during and shortly after Purkinje cell neurogenesis (gestational days 13-18) via oral intubation of pregnant dams. A second group received alcohol (2.5 g/kg/day) during early Purkinje cell differentiation (postnatal days 4-9) via artificial rearing of pups. The two alcohol treatment protocols were designed to match the cyclic daily blood alcohol profiles of the two groups as closely as possible. Pair-fed intubated controls, artificially reared gastrostomy controls, and normally reared ad lib/suckle controls were also evaluated. Mean peak blood alcohol concentrations (BACs) were 266 mg/dl for the intubated pregnant dams and 205 mg/dl for the pups exposed postnatally. Purkinje cell profiles were counted from single, 2-microns-thick midsaggital sections on postnatal day 10. Alcohol exposure during neurogenesis resulted in no significant change in Purkinje cell profile densities. Exposure during differentiation produced significant reductions in Purkinje cell profile densities, predominantly in the early maturing regions of the vermis (lobules I-IV and IX-X). These results indicate that Purkinje cells are more vulnerable to alcohol-induced population depletion during differentiation than during neurogenesis.     

Urokinase-type plasminogen activator regulates cranial neural crest cell migration in vitro

Proper migration and differentiation of neural crest (NC) cells are required for normal development of craniofacial structures, heart and great vessels, sensory and autonomic nervous systems, and other organs with vertebrate embryos. Serine-protease inhibitors reduce NC cell migration in vitro, suggesting the extracellular proteases are important mediators of NC cell migration. While plasminogen activator activity levels are high in NC cells relative to other embryonic tissue, its ability to regulate NC cell migration has not been specifically tested in vivo or in vitro through its ability to convert plasminogen to plasmin. Using a transfilter migration assay, NC cell migration was measured in the presence or absence of plasminogen. Our results showed that plasminogen significantly enhanced NC cell migration. This increase could not be attributed to differences in initial NC cell attachment or cytotoxicity and did not require a chemotactic gradient. The plasminogen-enhanced NC cell migration was blocked by aprotinin (a plasmin inhibitor) and was mimicked by the direct addition of plasmin to the NC cells, indicating that the plasminogen effect was mediated through plasmin generation. Furthermore, anticatalytic-uPA antibody blocked the plasminogen-enhanced NC cell migration showing that NC cell-associated uPA activity was required for this effect. Finally, decreasing NC-uPA activity by treating cells with transforming growth factor-Beta, also blocked the plasminogen-dependent increase in cell migration. These data show that in vitro, NC cell migration is regulated by NC-associated uPA activity suggesting that growth factor-regulation of this activity may play a major role in regulating NC cell migratory capacity in vivo.     

Stability and plasticity of neural crest patterning and branchial arch Hox code after extensive cephalic crest rotation

The extent to which the spatial organisation of craniofacial development is due to intrinsic properties of the neural crest is at present unclear. There is some experimental evidence supporting the concept of a prepattern established within crest while contiguous with the neural plate. In experiments in which the neural tube and premigratory crest are relocated within the branchial region, crest cells retain patterns of gene expression appropriate for their position of origin after migration into the branchial arches, resulting in skeletal abnormalities. But in apparent conflict with these findings, when crest is rerouted by late deletion of adjacent crest, infilling crest alters its pattern of gene expression to match its new location, and a normal facial skeleton results. In order to reconcile these findings thus identify processes of relevance to the course of normal development, we have performed a series of neural tube and crest rotations producing a more extensive reorganisation of cephalic crest than has been previously described. Lineage analysis using DiI labelling of crest derived from the rotated hindbrain reveals that crest does not migrate into the branchial arch it would have colonised in normal development, rather it simply populates the nearest available branchial arches. We also find that crest adjacent to the grafted region contributes to a greater number of branchial arches than it would in normal development, resulting in branchial arches containing mixed cell populations not occurring in normal development. We find that after exchange of first and third arch crest by rotation of r1-7, crest alters its expression of hoxa-2 and hoxa-3 to match its new location within the embryo resulting in the reestablishment of the normal branchial arch Hox code. A facial skeleton in which all the normal components are present, with some additional ectopic first arch structures, is formed in this situation. In contrast, when second and third arch crest are exchanged by rotation of r3 to 7, ectopic Hox gene expression is stable, resulting in the persistence of an abnormal branchial arch Hox code and extensive defects in the hyoid skeleton. We suggest that the intrinsic properties of crest have an effect on the spatial organisation of structures derived from the branchial arches, but that exposure to increasingly novel environments within the branchial region or "community effects" within mixed populations of cells can result in alterations to crest Hox code and morphogenetic fate. In both classes of operation we find that there is a tight link between the resulting branchial arch Hox code and a particular skeletal morphology.     

In vitro pattern formation during neurogenesis in neuroectodermal progenitor cells immortalized by p53-deficiency

In vitro neural differentiation was induced in a p53-deficient immortalized neuroectodermal progenitor cell line, NE-4C, by treatment with retinoic acid [K. Schlett and E. Madarász (1997) J. Neurosci. Res. 47, 405-416]. Rearrangement of nestin filaments was an early marker of neuron-formation. The increase in neurofilament protein content was accompanied by a decrease in the expression of nestin filaments in induced precursors. Cells with astroglial features appeared with a delay of 4-5 days compared to the appearence of neurons. Future neurons were sorted out from the substrate-attached population of apparently non-induced cells. The sorting out of future neurons resembled the separation of neural precursors in vivo. The continuous changes in the shape and also in the position of the cells resulted in the formation of characteristic morphological patterns. On the basis of morphological changes, five characteristic stages of in vitro neural differentiation were distinguished. The analysis of the morphological changes revealed that cell-to-cell interactions played an essential role in the cell fate decision made by induced precursors. Our observations indicate that the NE-4C cell line can serve as an in vitro model to investigate some early steps of neurogenesis.