Changes in cytosolic calcium, bleb formation, and cell death in neural crest cells treated with isotretinoin and 4-oxo-isotretinoin

Information regarding the cytopathologic mechanism of action of the retinoids [isotretinoin (IR) and 4-oxo-isotretinoin (4-OIR)] on neural crest cells (NCCs) in culture was sought. Those pathophysiologic alterations in cell metabolism studied were: cell blebbing (xieosis), free radical formation, cell viability, and cellular calcium homeostasis. Cells were treated with IR or 4-OIR in the presence of high (1.4 mM) and low (5.0 microM) levels of extracellular calcium ions. Recently developed techniques utilizing fluorescent molecular probes for calcium analyses, i.e., Fura 2AM, were used to study the effects of these drugs on the cytosolic calcium concentration of NCCs. The effects of IR and 4-OIR on NCC viability, [Ca++]int, were contrasted with the effects of certain sulfhydryl drugs (HgCl2, NEM, PCMBS) and calcium ionophores (ionomycin, A23187), agents known to perturb cell membranes, increase cytosolic calcium loads, and induce cell injury and subsequent cell death. Both retinoids were shown to induce an increase in the generation of superoxide radicals (SO) and increase the influx of calcium ions by the NCCs, thus increasing [Ca++]int by several hundred percent within 5 to 10 min. The liberation of SO was calcium dependent. These early effects were accompanied by an increase in cell blebbing activity. Also, a significant decrease in NCC viability was seen as early as 10 min after the addition of IR or 4-OIR to the incubation medium. 4-OIR proved to be the more potent of the two retinoids tested. The severity of these effects on NCC metabolism was dependent on medium calcium concentration with all changes being increased in the presence of the higher extracellular calcium levels. From the data presented it appears as though the retinoids cause a rapid elevation in cytosolic [Ca++]int possibly by purturbing the integrity of the cell membrane, denaturing membrane Ca-ATPase activity, or both. Retinoid-induced changes in membrane activity are evidenced by increased surface blebbing and superoxide formation. The prolonged elevation of intracellular [Ca++] may be directly related to depressed NCC viability and thus explain the known teratogenic effects of these drugs and their relationship to ectomesenchymal cell hypoplasia and craniofacial dysmorphogenesis.     

Effects of isotretinoin on the behavior of neural crest cells in vitro

Isotretinoin (13-cis-retinoic acid), an anti-acne medication, has been found to cause severe birth defects which affect the craniofacial elements, ear, heart, thymus, and central nervous system. Many of these structures receive contributions from the cranial neural crest. Here, we examine the possibility that these teratogenic effects are due to disturbances in neural crest development. Cranial and trunk neural crest explant cultures were exposed to different concentrations of isotretinoin and the cell morphology was monitored at daily intervals. Treated neural crest cells often became rounded or spindle shaped, separated from their neighbors, and frequently detached from the substrate or clumped together. In contrast, neural tube cells and cardiac fibroblasts were relatively unaffected by the drug. These results suggest that isotretinoin selectively affects neural crest cells by decreasing their cell-substratum adhesion.     

Invasive characteristics of neural crest cells in vitro

An investigation of the invasiveness of avian neural crest cells and neural crest-derived melanocytes through a human amniotic basement membrane (BM) was undertaken. Avian neural tube explants or derived melanocyte populations were seeded directly onto BMs in membrane invasion culture system (MICS) chambers for periods of 24, 48, and 72 h. In 36 experimental trials for each group, neither neural crest nor neural crest-derived melanocytes were observed to have invaded the BMs. In concert with these studies, coculturing of B16F10 murine melanoma cells with avian neural crest-derived melanocytes was performed in MICS chambers. Under these experimental conditions, the neural crest-derived melanocytes were able to successfully invade the BMs and to a greater extent than the B16F10 tumor cells. These data suggest that neural crest cells and neural crest-derived melanocytes do not have the ability to invade the BM alone; however, they can be induced to be invasive when cocultured in the presence of B16F10 cells. Alternatively, the B16F10 cells may create weaknesses within the BM that facilitate migration of the pigmented crest cells.     

Developmental effects of isotretinoin and 4-oxo-isotretinoin: the role of metabolism in teratogenicity

Previous observations have indicated that isotretinoin (IT), a drug in common use for therapy of cystic acne, is teratogenic in humans but possesses low embryotoxicity in pregnant mice, probably because of its shorter half-life and limited placental transfer in rodents. In human volunteers and patients, one major blood metabolite of IT is 4-oxo-isotretinoin (4-oxo-IT) which undergoes slower elimination than IT and may itself be a participant in teratogenesis. To investigate the problem of species differences displayed by IT and the role of its metabolism, embryotoxic effects of 4-oxo-IT were examined after its single or repeated intubations into pregnant ICR mice and compared with the effects of a similar regimen of IT. The two compounds were also tested for their relative ability to suppress chrondrogenesis in the in vitro cell and organ culture assays. We found that a single dose of 4-oxo-IT, 100 mg/kg, given on day 11 of gestation (plug day = day 0 of gestation) produced a moderate incidence of limb reduction defects and cleft palate (39% and 27% of surviving fetuses, respectively), while a dose of 150 mg/kg affected virtually every fetus. IT, on the other hand, produced no defects in fetuses exposed to similar dose levels. Repeated intubations with IT, however, resulted in increasing the frequencies of limb reduction defects and cleft palate to levels obtained after 4-oxo-IT administration. We found that a 3-hour interval between IT intubations was more effective in this regard than an 8-hour interval. Repeated IT intubations also uncovered sharper stage-dependency of limb and palatal defects than obtained otherwise.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neuronal differentiation of mouse neural crest cells in vitro

The purpose of the present study is to analyze the effect of serum or chick embryo extract (CEE) on the neuronal differentiation of the mouse neural crest cells. When the crest cells were cultured in the medium containing serum at low concentration (5% calf serum), neurite outgrowth was observed. The active outgrowth was detected at 3-4 days in culture. However, in the medium supplemented with 20% calf serum, no neurite appeared, and the crest cells remained fibroblast-like. The differentiation of adrenergic neurons was observed when the crest cells were cultured in the medium containing CEE along with serum.     

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.     

Coexpression of sensory and autonomic neurotransmitter traits by avian neural crest cells in vitro

This study shows that explants of quail neural crest cultured in a medium containing serum and chick embryo extract give rise to large numbers of cells expressing immunoreactivity for substance P (SP), a neuropeptide found in sensory neurons. These cells arise from cycling precursors, but do not appear to divide after expressing SP. The SP-positive cells in cranial neural crest cultures express both neurofilament and the Q211 antigen, but those in trunk cultures express only the Q211 antigen. In both cranial and trunk cultures, large subpopulations of the SP-positive cells express tyrosine hydroxylase and/or choline acetyltransferase, neurotransmitter markers characteristic of autonomic neurons. This finding argues against the idea that SP expression necessarily indicates commitment to the sensory neuron lineage. I further show that embryonic dorsal root ganglion (DRG) cells retain the ability to coexpress SP and tyrosine hydroxylase in vitro although to a lesser extent than do neural crest cells.     

Morphology and behaviour of neural crest cells of chick embryo in vitro

Summary Neural primordia of chick embryos were cultured for three days and the behaviour of migrating neural crest cells studied. Somite cells were used as a comparison. Crest cells were actively multipolar with narrow projections which extended and retracted rapidly, contrasting to the gradual extension of somite-cell lamellae. On losing cell contact, somite cells were also more directionally persistent. The rate of displacement of isolated crest cells was particularly low when calculated over a long time base. Both crest and somite cells were monolayered; contact paralysis occurred in somite cell collisions but was not ascertained for crest cells. However, crest cells in a population were far more directionally persistent than isolated cells. Contact duration between crest cells increased with time and they formed an open network. Eventually, retraction clumping occurred, initially and chiefly at the periphery of the crest outgrowth. Crest cells did not invade cultured embryonic mesenchymal or epithelial populations but endoderm underlapped them. No effects were observed on crest cells prior to direct contact. Substrate previously occupied by endoderm or ectoderm caused crest cells to flatten while substrate previously occupied by the neural tube caused them to round up and clump prematurely.     

Coexpression of sensory and autonomic neurotransmitter traits by avian neural crest cells in vitro.

This study shows that explants of quail neural crest cultured in a medium containing serum and chick embryo extract give rise to large numbers of cells expressing immunoreactivity for substance P (SP), a neuropeptide found in sensory neurons. These cells arise from cycling precursors, but do not appear to divide after expressing SP. The SP-positive cells in cranial neural crest cultures express both neurofilament and the Q211 antigen, but those in trunk cultures express only the Q211 antigen. In both cranial and trunk cultures, large subpopulations of the SP-positive cells express tyrosine hydroxylase and/or choline acetyltransferase, neurotransmitter markers characteristic of autonomic neurons. This finding argues against the idea that SP expression necessarily indicates commitment to the sensory neuron lineage. I further show that embryonic dorsal root ganglion (DRG) cells retain the ability to coexpress SP and tyrosine hydroxylase in vitro, although to a lesser extent than do neural crest cells.     

Ethanol induces the generation of reactive free radicals by neural crest cells in vitro

Developmental craniofacial anomalies related to the neural crest derived ectomesenchymal cell population are associated with fetal alcohol syndrome (FAS). Information regarding any potential relationship between ethanol, free radicals, and the viability, proliferation, etc., of isolated neural crest cells was sought. The hypersensitivity of neural crest cells to ethanol was observed. This drug severely depressed cell viability while simultaneously inducing the generation of such reactive oxygen intermediates (ROI) as superoxide, hydrogen peroxide, and hydroxyl anions. Addition of the free radical scavenging enzyme superoxide dismutase to the culture medium significantly reversed these effects of ethanol. The cytotoxicity of ethanol was further confirmed by the release of radiolabeled chromium (51Cr) from cells prelabeled prior to ethanol treatment. This effect was also depressed by the addition of superoxide dismutase. Interestingly, an assay for superoxide dismutase activity showed that neural crest cells may be devoid of this enzyme. The latter may help to explain the overt sensitivity of these cells to such a broad spectrum of teratogens, many of which can either dissociate directly into ROI, or cause the radicalization of biological structures and molecules. Plasmalemmal lipids (via lipid peroxidation) and DNA are at an especially high risk from uncontrolled ROI. Changes in neural crest cell surface morphology, i.e., loss of microvilli, formation of xeiotic blebs, as well as the "leakage" of radiolabeled Cr from prelabeled cells, would seem to show that ethanol, as a result of induced free radical formation, alters the physiology and biochemistry of the cell membrane. These findings however, should not exclude other potential sites for ETOH-induced cell injury related to free radicals, especially the nuclei (DNA), mitochondria, organelle membranes, and the cytoskeleton.     

Immortalization and controlled in vitro differentiation of murine multipotent neural crest stem cells

To isolate mouse neural crest stem cells, we have generated a rat monoclonal antibody to murine neurotrophin receptor (p75). We have immortalized p75⁺ murine neural crest cells by expression of v-myc, and have isolated several clonal cell lines. These lines can be maintained in an undifferentiated state, or induced to differentiate by changing the culture conditions. One of these cell lines, MONC-1, is capable of generating peripheral neurons, glia, and melanocytic cells. Importantly, most individual MONC-1 cells are multipotent when analyzed at clonal density. The neurons that differentiate under standard conditions have an autonomic-like phenotype, but under different conditions can express markers of other peripheral neuronal lineages. These lines therefore exhibit a similar differentiation potential as their normal counterparts. Furthermore, they can be genetically modified or generated from mice of different genetic backgrounds, providing a useful tool for molecular studies of neural crest development. © 1997 John Wiley & Sons, Inc. J Neuroblol 32 : 722–746, 1997     

Cellular fibronectin promotes adrenergic differentiation of quail neural crest cells in vitro

We have investigated the interaction of cellular fibronectin (CFN) with cultured quail neural crest cells and its possible role in crest cell migration and differentiation. In vitro, quail neural crest cells from the trunk region differentiate into at least two morphologically recognizable cell types, melanocytes and adrenergic nerve cells. The latter often aggregate spontaneously into ganglia-like structures. We found that neither melanocytes nor adrenergic nerve cells synthesize CFN. However, both cell types readily interacted with exogenous CFN: Melanocytes removed CFN from the substratum and accumulated it in an aggegated form on their upper cell surface, whereas unpigmented cells migrated on the CFN substratum, often rearranging it into a fibrillar network. The adsorption of CFN by melanocytes was apparently without further consequences. However, catecholamine-positive cells were substantially increased after treatment with exogeneous fibronectin. The stimulation of adrenergic differentiation of neural crest cells is the first evidence for a positive regulatory role of fibronectin in differentiation.     

Melanocyte-stimulating hormone affects melanogenic differentiation of quail neural crest cells in vitro

Quail neural crest cells were treated in vitro with alpha-melanocyte-stimulating hormone (alpha-MSH) or dibutyryl cyclic AMP (dbcAMP) plus theophylline. These treatments increased the proportion of melanocytes to total cells in crest cell outgrowth cultures. Pigmentation of neural crest cell clusters proceeded more rapidly when cultures were treated with alpha-MSH or dbcAMP plus theophylline than when untreated. In clonal cell cultures, the proportion of pigmented colonies to total colonies was increased by MSH treatment. From these results, MSH seems not only to accelerate melanogenic differentiation but also to affect the state of commitment of neural crest cells to melanogenic differentiation in vitro, and this action of MSH appears to be mediated by cAMP.     

A mammalian in vitro model to study gangliogenesis from neural crest cells

In spite of considerable advances towards understanding lineages derived from neural crest cells using amphibian and avian embryos, the molecular mechanisms involved in the formation of mammalian peripheral ganglia remain largely unknown, mainly because of the lack of experimental systems that will allow their in vitro manipulation. Here, we present a novel mammalian in vitro model permitting to study gangliogenesis from neural crest cells. This model allowed us to manipulate molecules involved in cell-cell interactions. Our data are in favour of the existence of a hierarchy among adhesion molecules.     

Migration and proliferation of cultured neural crest cells in W mutant neural crest chimeras.

Chimeric mice, generated by aggregating preimplantation embryos, have been instrumental in the study of the development of coat color patterns in mammals. This approach, however, does not allow for direct experimental manipulation of the neural crest cells, which are the precursors of melanoblasts. We have devised a system that allows assessment of the developmental potential and migration of neural crest cells in vivo following their experimental manipulation in vitro. Cultured C57Bl/6 neural crest cells were microinjected in utero into neurulating Balb/c or W embryos and shown to contribute efficiently to pigmentation in the host animal. The resulting neural crest chimeras showed, however, different coat pigmentation patterns depending on the genotype of the host embryo. Whereas Balb/c neural crest chimeras showed very limited donor cell pigment contribution, restricted largely to the head, W mutant chimeras displayed extensive pigmentation throughout, often exceeding 50% of the coat. In contrast to Balb/c chimeras, where the donor melanoblasts appeared to have migrated primarily in the characteristic dorsoventral direction, in W mutants the injected cells appeared to migrate in the longitudinal as well as the dorsoventral direction, as if the cells were spreading through an empty space. This is consistent with the absence of a functional endogenous melanoblast population in W mutants, in contrast to Balb/c mice, which contain a full complement of melanocytes. Our results suggest that the W mutation disturbs migration and/or proliferation of endogenous melanoblasts. In order to obtain information on clonal size and extent of intermingling of donor cells, two genetically marked neural crest cell populations were mixed and coinjected into W embryos. In half of the tricolored chimeras, no co-localization of donor crest cells was observed, while, in the other half, a fine intermingling of donor-derived colors had occurred. These results are consistent with the hypothesis that pigmented areas in the chimeras can be derived from extensive proliferation of a few donor clones, which were able to colonize large territories in the host embryo. We have also analyzed the development of pigmentation in neural crest cultures in vitro, and found that neural tubes explanted from embryos carrying wt or weak W alleles produced pigmented melanocytes while more severe W genotypes were associated with deficient pigment formation in vitro.     

Differential development of cholinergic neurons from cranial and trunk neural crest cells in vitro

Several studies have suggested that the development of cholinergic properties in cranial parasympathetic neurons is determined by these cells' axial level of origin in the neural crest. All cranial parasympathetic neurons normally derive from cranial neural crest. Trunk neural crest cells give rise to sympathetic neurons, most of which are noradrenergic. To determine if there is an intrinsic difference in the ability of cranial and trunk neural crest cells to form cholinergic neurons, we have compared the development of choline acetyltransferase (ChAT)-immunoreactive cells in explants of quail cranial and trunk neural crest in vitro. Both cranial and trunk neural crest explants gave rise to ChAT-immunoreactive cells in vitro. In both types of cultures, some of the ChAT-positive cells also expressed immunoreactivity for the catecholamine synthetic enzyme tyrosine hydroxylase. However, several differences were seen between cranial and trunk cultures. First, ChAT-immunoreactive cells appeared two days earlier in cranial than in trunk cultures. Second, cranial cultures contained a higher proportion of ChAT-immunoreactive cells. Finally, a subpopulation of the ChAT-immunoreactive cells in cranial cultures exhibited neuronal traits, including neurofilament immunoreactivity. In contrast, neurofilament-immunoreactive cells were not seen in trunk cultures. These results suggest that premigratory cranial and trunk neural crest cells differ in their ability to form cholinergic neurons.