Differentiation of avian neural crest cells in vitro: Absence of a developmental bias toward melanogenesis

Summary Neural crest cells from quail embryos grown in standard culture dishes differentiate almost entirely into melanocytes within 4 or 5 days when chick embryo extract (CEE) or occasional lots of fetal calf serum (FCS) are included in the medium. Gel fractionation showed that the pigment inducing factor(s) present in these media is of high molecular weight (> 400 K daltons). In the absence of CEE, the neural tube can also stimulate melanocyte differentiation. Culture medium supplemented by selected lots of FCS permits crest cell proliferation but little overt differentiation after up to 2 weeks in culture if the neural tube is removed within 18 h of explantation in vitro. Subsequent addition of CEE to such cultures promotes complete melanocyte differentiation. Crest cells from White leghorn chick embryos also differentiate into melanocytes in the presence of CEE, but do not survive well in its absence. Melanocyte differentiation of crest cells from both quail and chick embryos can by suppressed by culturing under a dialysis membrane, even in the presence of the neural tube and CEE, but neuronal differentiation appears greatly enhanced.     

Differentiation of reptilian neural crest cells in Vitro

An attempt was made to culture neural crest cells of the turtle embryo in vitro. Trunk neural tubes from the St. 9/10 embryos were explanted in culture dishes. The developmental potency of the turtle neural crest cells in vitro was shown to be essentially similar to that of avian neural crest cells, although they seem to be more sensitive to melanocyte-stimulating hormone (MSH) stimulation. We describe conditions under which explanted neural tube gives rise to neural crest cells that differentiate into neuronal cells and melanocytes. The potency of melanocyte differentiation was, found to vary according to the concentration of fetal bovine serum (FBS, from 5 to 20%). Melanization of neural crest cells cultured in the medium containing FBS and α-MSH was more extensive than those cultured with FBS alone, combinations of FBS and chick embryo extract, or turtle embryo extract. These culture conditions seem to be useful for the study of the developmental potency of the neural crest cells as well as for investigating local environmental factors.     

Analysis of developmentally homogeneous neural crest cell populations in vitro: I. Formation,morphology and differentiative behavior

When early embryonic quail neural tubes are dissected free from surrounding tissues and placed in culture, small stellate neural crest cells usually migrate from the explant onto the substratum. This outgrowth has been reported to consist of a mixture of cells, some of which undergo melanogenesis, while the rest remain unpigmented. We have, in contrast to earlier observations, obtained a spatial separation of the two phenotypes. In these cultures the primary outgrowth of migrating cells remained almost free of pigment-forming cells, whereas small spherical clusters containing several hundred pigment-forming cells appeared on the explanted neural tubes. Whether the clusters remained with the tube explants or were subcultured, all cluster cells differentiated into melanocytes. Prior to melanogenesis, the appearance of the cultured cells from a cluster was indistinguishable from the cells in the outgrowth. The clusters provide a source of neural crest cells, that (1) can be easily obtained in comparatively large numbers, (2) is not contaminated with any other cell type, (3) can be isolated before the onset of differentiation, and (4) is developmentally homogeneous. Thus, the cluster population is well suited for many types of experiments, such as the identification of specific environmental factors that might control neural crest cell differentiation.     

Analysis of developmentally homogeneous neural crest cell populations in vitro. IV. Cell proliferation and synthesis of glycosaminoglycans

Glycosaminoglycans (GAG) have been implicated as regulators of morphogenesis and differentiation. We have cultured two different homogeneous populations of quail trunk neural crest cells with different predictable phenotypes after equivalent times in culture, and used these populations to distinguish changes in GAG synthesis before and after cell differentiation from changes that depend on time in culture, and that thus may reflect a non-specific response to in vitro culture conditions. Cells derived from the outgrowths surrounding explanted neural tubes remained mainly undifferentiated. These crest cells showed a decrease in [³H]glucosamine incorporation into total GAG with time in culture. A similar decrease in total GAG, and, in addition, a slight decrease in the proportion of hyaluronic acid (HA) was observed in cultures of cluster-derived cells that homogeneously differentiated into melanocytes. Putative mouse neural crest cells that did not form melanin under the present culture conditions showed, similarly to the quail neural crest cells, a high incorporation of [³H]glucosamine into HA relative to sulfated GAG. The proportion of HA did not decrease with time in culture in these mouse crest cells. When pigment granules appeared in avian crest cells, the proliferation rate decreased drastically, whereas the proliferation rate of cells that did not form pigment granules remained constant. The results indicate that time in culture rather than either a differentiation per se or a change in the rate of proliferation, is largely responsible for the observed changes in GAG synthesis.     

Substrate dependence of cell migration from explanted neural tubes in vitro

Summary Embryonic chick neural tubes containing neural crest cells were cultured in vitro on tissue culture plastic and collagen. Two parameters, the time of onset of cell migration from the neural tube and the rate of movement of the cell front away from the neural tube explant, were determined. On collagen, cell migration consistently began after four to six h in vitro, about five h prior to the onset of cell migration on tissue culture plastic. The identity of the migrating cells as neural crest cells is established by their eventual differentiation into melanocytes. Ablation experiments reveal that collagen also causes the early onset of migration of cells not of neural crest origin. These results provide in vitro support for the idea that extracellular materials may alter cell migratory behaviour in morphogenesis.Supported by PHS grant HD-05395 to Dr. James A. Weston and NIH Predoctoral Research Fellowship GM-47392 to Gerald D. Maxwell. The author thanks John Pintar for his permission to quote unpublished observations on the neural crest films and for helpful discussion, and Dr. Peter H. von Hippel for the gift of icthyocol     

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.     

Cell lineages in peripheral nervous system ontogeny: medium-induced modulation of neuronal phenotypic expression in neural crest cell cultures

Neural crest, taken from cephalic and trunk levels of quail embryos, was grown in vitro in conventional tissue culture medium (Dulbecco's modified Eagle's medium containing 15% fetal calf serum and either 2 or 15% chick embryo extract (CEE] or in a chemically defined serum- and CEE-free medium. Depending on the conditions employed, different types of neuronal or neuronlike cells developed in the cultures. Thus, in medium containing 15% CEE, adrenergic cells (identified by tyrosine hydroxylase immunoreactivity and catecholamine histofluorescence) emerged after 5-6 days. These cells lacked tetanus toxin binding sites and did not react with an antibody directed against 70-kDa neurofilament protein. In the fully defined medium, a neuronal cell type exhibiting neurofilament and substance P (SP) immunoreactivity differentiated from noncycling precursors within 1 or 2 days of culture. If serum was added to the medium, the neurites disintegrated and the neuronal cells ultimately died. By sequentially culturing neural crest, first in the wholly synthetic medium for 1-3 days and then in the conventional medium supplemented with serum and 15% CEE, the disappearance of the SP-positive neurons was followed, several days later, by the emergence of adrenergic cells. The majority of these cells and/or their precursors were found to undergo cell division in culture. We conclude that the cells expressing the adrenergic phenotype (characteristic of the sympathetic nervous system) and those displaying SP immunoreactivity, comparable to a category of neurons in dorsal root and cranial sensory ganglia, derive from distinct sets of precursors. Our results reinforce the contention, deduced from in ovo transplantation experiments (see N. M. Le Douarin, (1984) In Cellular and Molecular Biology of Neuronal Development (I. Black, Ed.), pp. 3-28. Plenum, New York), that at least two lineages, from which sensory and autonomic cell types are derived respectively, are segregated early during neural crest ontogeny and have extremely different survival and trophic requirements.     

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.     

Plasminogen activator activity is associated with neural crest cell motility in tissue culture

We have examined the possibility that proteases such as plasminogen activator (PA) contribute to the extraordinary motile capability of neural crest cells. We show that trunk neural crest cells that migrate from isolated neural tubes in vitro produce PA and that the level of cell-associated PA increases dramatically after 8 days in culture. This increase is not the result of differentiation or time in culture, because neural crest cell clusters that form on top of the neural tube and differentiate into pigment cells but are immotile produce very low levels of PA. If these clusters are removed from the neural tube and replated on a plastic substratum where they migrate, the level of PA associated with the cells increases dramatically, suggesting that PA production is associated with motility. Inhibitors of PA/plasmin activity significantly reduce neural crest cell motility in vitro, further supporting the idea that proteases are important in neural crest cell migration.     

Analysis of the development of cellular subsets present in the neural crest using cell sorting and cell culture

We have tested the hypothesis that developmentally significant cellular subsets are present in the early stages of neural crest ontogenesis. Cultured quail trunk neural crest cells probed with the monoclonal antibodies HNK-1 and R24 exhibited heterogeneous staining patterns. Fluorescence-activated cell sorting was used to isolate the HNK-1+ and HNK-1- cell populations at 2 days in vitro. When these cell populations were cultured, the HNK-1+ sorted cells differentiated into melanocytes, unpigmented cells, and numerous catecholamine-positive (CA+) cells. In contrast, the HNK-1- sorted cells gave rise to melanocytes and unpigmented cells, but few, if any, CA+ cells. When neural crest cells at 2 days in vitro were labeled with R24 and sorted, both the R24+ the R24- sorted cell populations produced numerous CA+ cell, melanocytes, and unpigmented cells. These results provide evidence for the existence of developmental preferences in some subsets of neural crest cells early in embryogenesis.     

Explanted Fish Neural Tubes Give Rise to Differentiating Neural Crest Cells

Neural tubes were explanted from the trunk of various embryonic stages of three teleost fish, Xiphophorus maculatus (platyfish), X. helleri (swordtail), and Oryzias latipes (Japanese medaka) with the aim to obtain in vitro differentiating neural crest cells. Outgrowth of cells was observed immediately after attachment of the explants on dishes coated with fibronectin. The outgrowing cells stained with the HNK-1 monoclonal antibody indicating that they were neural crest cells. Maximum cell outgrowth was obtained from explants of stage 9 of Xiphophorus and 19 of medaka, i.e., from stages characteristic of maximal neural crest cell segregation, and by the use of Leibovitz's (L-15) medium supplemented with 20% FBS. In this medium cells survived for more than two weeks; M199 also gave satisfactory results but DMEM allowed only poor cell growth and survival. Neuronal cells could be observed in all cultures after 48 hr, in some medaka cultures these cells were mixed with pigment cells but homogeneous pigment cell cultures were also observed. This in vitro system will be invaluable for the study of the developmental potential of fish neural crest cells and the contributions of extrinsic factors in neural crest cell fate.     

Molecular identification of distinct neurogenic and melanogenic neural crest sublineages

Clonal and lineage analyses have demonstrated that although some neural crest cells have the ability to generate multiple cell types and display self-renewal ability, other crest cells generate a single or limited repertoire of cell types. However, it is not yet clear when, and in what order, crest cells become specified to adopt a particular fate. We report that the receptor tyrosine kinases TrkC and C-Kit are expressed by distinct neural crest subpopulations in vitro. We then analyzed the lineages of individual receptor-expressing crest cells and found that TrkC-expressing cells that have just emerged from the neural tube give rise to clones containing neurons or glial cells, or both, but never produce melanocytes. A short time later, TrkC-expressing cells only generate pure neuronal clones. By contrast, from their earliest appearance in neural tube outgrowths, C-Kit-expressing cells invariably give rise to clones containing only melanocytes. Our results directly demonstrate that distinct neurogenic and melanogenic sublineages diverge before or soon after crest cells emerge from the neural tube, that fate-restricted precursors are present in nascent neural crest populations and that these sublineages can be distinguished by their cell type-specific expression of receptor tyrosine kinases.     

Transforming growth factor-beta alters differentiation in cultures of avian neural crest-derived cells: effects on cell morphology, proliferation, fibronectin expression, and melanogenesis.

Neural crest cell differentiation is responsive to a variety of extrinsic signals that include extracellular matrix (ECM) molecules and growth factors. Transforming growth factor-beta (TGF-beta) has diverse, cell type-specific effects, many of which involve regulation of synthesis of ECM molecules and their cell surface receptors. We are studying both separate and potentially interrelated influences of ECM and growth factors on crest differentiation and report here that TGF-beta alters several aspects of crest cell behavior in vitro. Clusters of quail neural crest cells were cultured in the presence and absence of 400 pM TGF-beta 1 and examined at 1, 3, and 5 days. When examined at 5 days, there was a dramatic decrease in the number of melanocytes in treated cultures, regardless of the onset or duration of TGF-beta treatment. With continuous TGF-beta treatment, or with treatment only during crest cluster formation on explanted neural tubes, many cells increased in area, becoming extremely flat. These changes were evident beginning on Day 3. While quantitative analyses of video images documented the size increase, several aspects of motility were relatively unchanged. Synthesis of fibronectin (FN) by approximately 11% of cells on Day 3 and 31% of cells on Day 5 was demonstrated by immunocytochemistry and was associated with a sixfold increase in FN mRNA by Day 5. Experiments which correlated FN immunoreactivity with incorporation of bromodeoxyuridine suggested that the population of large, flat, FN-positive cells did not proliferate selectively and that there was a slower rate of proliferation in TGF-beta-treated cultures than in untreated cultures. The large FN-immunoreactive cells resemble cells derived from cephalic neural crest and raise interesting questions concerning potential roles for TGF-beta in regulating crest differentiation in vivo.     

A clonal approach to the problem of neural crest determination.

A fundamental question regarding neural crest development is the possible pluripotential nature of this embryonic tissue. As a first step in examining this problem, clonal techniques are used to produce homogeneous populations of crest cells. Primary cultures of these cells are obtained by explanting neural tubes from Japanese quail in vitro and allowing crest cells to migrate away. The explant is removed, the outgrowth is isolated, dissociated with trypsin, and the cells plated at clonal density. Colonies derived in this manner fall into the following categories: all cells of the colony pigmented; none of the cells pigmented; and some of the cells pigmented, the remainder unpigmented. Pigmented colonies generally arise from small, round cells whereas the non-pigmented colonies usually originate from large, flattened polymorphous cells. Differentiation of melanocytes does not preclude their continued proliferation. The pigment phenotype, in addition, is stable through at least 25 generations. That the mixed colonies, in fact, are clonally derived is shown by physically isolating single cells. The identity of the non-pigment cells was not established in the present work. A possible neural fate is suggested, however, since nerve-like cells develop after the petri plates become overgrown. Neural clones did not form even though nerve growth factor activity is present as a normal constituent of the culture medium and was added as a supplement in some instances. These techniques permit the preparation of large, homogeneous populations of neural crest cells and afford an opportunity to examine aspects of crest determination heretofore impossible to study.     

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.     

Substratum effects on cell dispersal, morphology, and differentiation in cultures of avian neural crest cells

Adhesive extracellular matrix (ECM) molecules appear to play roles in the migration of neural crest cells, and may also provide cues for differentiation of these cells into a variety of phenotypes. We are studying the influences of specific ECM components on crest differentiation at the levels of both individual cells and cell populations. We report here that the glycoproteins fibronectin and laminin differentially affect melanogenesis in cultures of avian neural crest-derived cells. Clusters of neural crest cells were allowed to form on explanted neural tubes for 24 and 48 hr, and then subcultured on uncoated glass coverslips or coverslips coated with fibronectin or laminin. The morphology of cells varied on the three substrata, as did patterns of cell dispersal. Crest cells dispersed most rapidly and extensively on fibronectin. In contrast, cells on laminin dispersed initially, but then assumed a stellate morphology and rapidly formed small aggregates. Cell dispersal was minimal on glass substrata, resulting in a uniformly dense distribution. These patterns of dispersal were similar in subcultures of both 24- and 48-hr clusters, although dispersal of cells from older clusters was less extensive. The rate and extent of melanogenesis correlated with patterns of cell dispersal. Cell from 24-hr clusters underwent melanogenesis significantly more slowly on fibronectin than on the other two substrata. Pigment cells began to differentiate by 2 days of subculture in the cell aggregates on laminin and in the dense centers of cultures on untreated glass. By 5 days, there was significantly more melanogenesis in cultures on laminin and glass than on fibronectin substrata. Melanogenesis in cultures of 48-hr clusters was more rapid and extensive on control (glass) substrata than on fibronectin or laminin, correlating with reduced cell dispersal. We conclude that fibronectin and laminin, which are found along neural crest migratory pathways in vivo, can affect melanogenesis in vitro by regulating patterns of cell dispersal.     

Establishment by an original single-cell cloning method and characterization of an immortal mouse melanoblast cell line (NCCmelb4)

We devised a unique new single-cell cloning method which uses microscope cover glasses and established a melanoblast cell line derived from mouse neural crest cells. A microscope cover glass was nicked and broken into small pieces and put on a dish. Culture medium and a suspension of 20-30 cells/ml were dropped in the dish. After 1-3 d, a piece of glass to which only one cell was adhered was picked up and transferred to another dish containing culture medium. The greatest advantage of this method is that the derivation of a colony from a single cell can be directly confirmed by microscopy and there is no risk of migratory cells being contaminated by other colonies. Using this single-cell cloning method, in this study we established a cell line derived from a neural crest cell line (NCC-S4.1) and designated it as NCCmelb4. When the culture medium was supplemented with stem cell factor (SCF) alone, NCCmelb4 cells were KIT-positive and tyrosinase-negative melanocyte precursors; they remained at an immature and undifferentiated stage. When the medium was supplemented with phorbol 12-o-tetradecanoyl-13-acetate (TPA) + cholera toxin (CT), the cell morphology changed and became L-3,4-dihydroxyphenylalanine (DOPA)-positive. This observation indicates that the NCCmelb4 cells are capable of further differentiation with suitable stimulation. NCCmelb4 cells derived from the mouse neural crest has characteristics of melanocyte precursors (melanoblasts), and is a cell line which can be utilized to study differentiation-inducing factors and growth factors without the effects of feeder cells.     

A low-serum medium with BSA and ferrous sulfate for the production of tissue plasminogen activator by human embryo lung cells

A low-serum medium containing bovine serum albumin (BSA) was investigated with respect to the growth of and tissue plasminogen activator (TPA) production by human embryo lung (HEL) cells on microcarrier beads and in collagen gel. BSA and ferrous sulfate were chosen as substitutes for fetal calf serum (FCS) through a simple screening test involving many substances. The growth promoting effects of BSA and ferrous sulfate were independent of each other and from the FCS concentration. Though BSA inhibited initial cell attachment to the carrier surface, it did promote the growth of cells attached to microcarrier beads. Cells grown on microcarrier beads in the low-serum medium containing BSA, ferrous sulfate and 3% FCS produced an amount of TPA similar to that produced by ones grown in the 10% FCS medium. Although cells on the dish surface did not grow at all on serum-free media containing BSA and ferrous sulfate, cells in the collagen gel were able to grow slightly on the serum-free medium. Cells grown on the low-serum medium in collagen gel produced more TPA over a long period than those in the microcarrier beads using the low-serum medium. The optimum concentration of proteose peptone in the TPA production medium for the collagen gel culture was similar to that for the dish surface culture.     

Gene transfer and expression studies in cultured avian neural crest cells differentiating into melanocytes

Neural crest cells obtained from explanted neural tubes take up, express, and retain exogenous DNA applied by the CaPO4 co-precipitation method during their differentiation into melanocytes. High efficiencies of gene transfer were obtained with both supercoiled DNA and intact phage particles; linear DNA or DNA from the phage yielded very low efficiencies. There is some evidence that transferred gene expression is differentiation dependent. The system should be useful for studies concerned with the analysis of cell developmental genes and their regulatory elements.     

Phenotypic dissections of the Blastocladiella emersonii zoospore's developmental choice

A developmentally homogeneous neural crest cell population has been used to assay the role of environmental factors in regulating crest cell differentiation. If cultured on tissue culture plastic, virtually all of the cells of this population differentiate into melanocytes. In contrast, when these cells are cultured for 3 or more days on substrata “conditioned” by somite fibroblasts, the proportion of cells undergoing melanogenesis decreased and the proportion expressing formaldehyde-induced fluorescence (FIF), characteristic of catecholamine-containing cells, increased. For a limited period of culture on somite-conditioned substrata, some cells in the population exhibit both pigment granules and fluorescence. Collagen-coated substrata decreased the number of cells that formed pigment but did not stimulate FIF. In contrast, optimum doses of exogenous cellular fibronectin mimicked the effect of somite-conditioned substrata, suppressing melanogenesis and promoting FIF. Glycosaminoglycan-derivatized substrata (i.e., hyaluronic acid, various chondroitin sulfate preparations, and heparin) did not alter the differentiative homogeneity of the cultured crest cell populations. The choice and expression of phenotype by some members of a cultured crest cell population can, therefore, be affected by environmental stimuli provided in the form of certain substrate-attached macromolecules. We suggest that optimal concentrations of some extracellular matrix components produced by embryonic tissue and normally encountered by migrating crest cells may elicit the expression of FIF in crest cells that would otherwise follow a different developmental pathway.