Clonal analysis of quail neural crest cells: They are pluripotent and differentiate in vitro in the absence of noncrest cells

To determine if neural crest cells are pluripotent and establish whether differentiation occurs in the absence of noncrest cells, a cell culture method was devised in which differentiation could be examined in clones derived from single, isolated neural crest cells. Single neural crest cells, which were isolated before the onset of in vivo migration, gave rise to three types of clones: pigmented, unpigmented, and mixed. Pigmented clones consisted of melanocytes only, whereas some unpigmented cells in mixed and unpigmented clones contained catecholamines, identifying them as adrenergic cells. Extracellular matrix derived from quail somite or chick skin fibroblast cultures stimulated adrenergic differentiation and axon formation. These results demonstrate for the first time the existence of pluripotent quail neural crest cells that give rise to at least two progeny, melanocytes and neuronal cells. They also suggest that continuous direct interactions with noncrest cells are not required for the differentiation of these two cell types. However, components of the extracellular matrix derived from noncrest cells may play an important role in expression of the adrenergic phenotype.     

Possible involvement of reorganization of actin filaments, induced by tumor-promoting phorbol esters, in changes in colony shape and enhancement of proliferation of cultured epithelial cells

Tumor promoters are known to induce reorganization of actin, morphological changes and enhancement of proliferation of epidermal cells in vivo. In this study, we have examined the effects of tumor promoters on these events to clarify the role played by the organization of actin filaments in the regulation of the shape and growth of colonies of epithelial cells in culture. Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) caused a change in the shape of colonies of FL and Madin-Darby canine kidney (MDCK) cells within 6 hr. Changes in the shape of colonies were consistent with the morphological change of individual cells and the dissociation of groups of cells in the colonies. Addition of TPA also caused reorganization of actin filaments after 2 hr, and it caused enhancement of proliferation of FL and MDCK cells after 48 hr but did not cause any such changes in KB cells. However, the binding affinities of 4 beta-phorbol 12,13-dibutyrate (PDBu) to FL and MDCK cells were similar to that of PDBu to KB cells. Related tumor promoters such as phorbol 12,13 didecanoate (PDD) and mezerein caused effects similar to those caused by TPA. In contrast, nontumor promoting phorbol esters, such as 4 alpha-PDD and phorbol, had no effect. Cyclic AMP blocked the TPA-induced changes in FL and MDCK cells. These results suggest that TPA-induced reorganization of actin filaments which can be inhibited by cyclic AMP results in changes in the shape of colonies and enhancement of proliferation.     

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.     

Changes in the migratory properties of neural crest and early crest-derived cells in vivo following treatment with a phorbol ester drug

In previous work, we found that the phorbol ester drug 12-O-tetradecanoyl phorbol acetate (TPA) reversed the developmental restriction of melanogenesis that normally occurs in neural crest-derived Schwann cell precursors around embryonic Day 5 of quail development. That is, TPA treatment of dorsal root ganglia (DRG) from 7-day quail embryos caused Schwann cell precursors to regain the ability to give rise to melanocytes. In this paper, we examine other long-term effects of TPA on the differentiative and migratory properties of neural crest and crest-derived DRG cells, using heterospecific grafting methods. We report that TPA treatment in culture increased the extent of cell migration following grafting into host embryos, including some ectopic migration into the central nervous system and other locations. TPA did not, however, seem to change the fate of these crest-derived cells, except that some DRG cells underwent pigmentation, as had been observed previously. Interestingly, graft cells associated with peripheral nerves were found to be exclusively unpigmented, whereas graft cells found in all other locations, including the central nervous system, were both pigmented and unpigmented. This suggests that peripheral nerves may act in a fashion antagonistic to the effects of TPA. These findings are consistent with the notion that TPA treatment causes early crest-derived cells to regain developmental properties lost with developmental age.     

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.     

Phorbol-ester stimulated lysis of weak and nonspecific target cells by cytotoxic T lymphocytes

We have investigated the effects of short-term incubation of cloned and in vivo-produced cytotoxic T lymphocytes (CTL) with phorbol esters on their lytic activity against weak and nonspecific targets. These experiments demonstrate that 4 beta-phorbol-12-myristate, 13-acetate (PMA), 4 beta-phorbol-12,13-dibutyrate, and 4 beta-phorbol-12,13-didecanoate, but not the 4 alpha-phorbol-12,13 didecanoate esters stimulate the lytic apparatus. The stimulation is specific for the CTL rather than the target and appears to be nearly instantaneous in action. This rapid stimulation of the CTL lytic process is consistent with previously reported effects of phorbol esters associated with T cell activation in other functional assays.     

Effect of lectins and sugars on primary sperm attachment in the horseshoe crab, Limulus polyphemus L

The in vitro differentiation of quail neural crest cells into serotoninergic neurons is reported. Serotoninergic neurons were identified by two independent methods, formaldehyde-induced histofluorescence and indirect staining with antiserotonin antibodies. Serotonin-positive cells first appeared on the third day in culture, simultaneously, or slightly prior to the first pigmented cells and adrenergic neurons. Comparable numbers of serotoninergic cells were found in crest cell cultures derived from vagal, thoracic/upper lumbar, and lumbosacral levels of the neuraxis. The neural crest origin of the serotonin neurons was further corroborated by the demonstration that cultures of somites, notochords, and neural tubes (three tissues adjacent to the neural crest and thus the most likely contaminants of crest cell cultures) did not contain serotonin-producing cells, and that mast cells were absent in crest cell cultures. The identification of serotoninergic neurons in quail neural crest cell cultures makes an important addition to the number of neural crest derivatives that are capable of differentiating in culture. Furthermore, it suggests that the in vitro culture system will prove a valid approach to the elucidation of the cellular and molecular mechanisms that govern neural crest cell differentiation.     

In vitro clonal analysis of quail cardiac neural crest development

The developmental potentials of cardiac neural crest cells were investigated by in vitro clonal analysis. Five morphologically distinct types of clones were observed: (1) "pigmented" clones contained melanocytes only; (2) "mixed" clones consisted of pigmented and unpigmented cells; (3) "unpigmented dense" clones consisted of flattened, closely aligned unpigmented cells; (4) "unpigmented loose" clones consisted of a few loosely arranged, flattened cells; and (5) "unpigmented large" clones included a large number of small, stellate cells that were highly proliferative. The binding patterns of antibodies against lineage-specific markers showed that cells in the different clones expressed characteristic phenotypes. The following phenotypes were expressed in addition to pigment cells: smooth muscle cells, connective tissue cells, chondrocytes, and cells in the sensory neuron lineage. Mixed clones expressed all five phenotypes. Unpigmented dense clones contained smooth muscle cells, connective tissue cells, chondrocytes, and sensory neurons. Unpigmented loose clones exclusively consisted of smooth muscle cells, whereas unpigmented large clones contained chondrocytes and sensory neuron precursors. Based on these results, the following conclusions can be drawn: (1) Pigmented and unpigmented loose clones are most likely formed by precursors that are committed to the melanogenic and myogenic cell lineages, respectively. (2) Mixed and unpigmented dense clones are derived from pluripotent cells with the capacity to give rise to four or five phenotypes. (3) Unpigmented large clones originate from progenitor cells that appear to have a partially restricted developmental potential, that is, these cells are capable of generating two phenotypes in clonal cultures. Thus, the data indicate that the early migratory cardiac neural crest is a heterogeneous population of cells, consisting of pluripotent cells, cells with a partially restricted developmental potential, and cells committed to a particular cell lineage.     

Pigment patterns in neural crest chimeras constructed from quail and guinea fowl embryos

The pattern of pigmentation in bird embryos is determined by the spatial organization of melanocyte differentiation. Some of the results from recent, neural crest transplantation experiments support a model based on a prepattern in the feathers; others could be interpreted in terms of a nonspecific pattern resulting from a failure of the crest cells to read the positional values in another species. To distinguish between these possibilities, the crucial test is to construct chimeras from two species with different pigment patterns. We have examined the wing plumage of quail and guinea fowl embryos. The quail has a characteristic pattern of pigmented and unpigmented feather papillae, whereas the guinea fowl shows uniform pigmentation. Chimeras were constructed by grafting wing buds isotopically between embryos. The wing buds were transplanted before they had become invaded by neural crest cells. Quail wing buds grafted to the guinea fowl developed, in most cases, a pigment pattern resembling that of the quail and not that of the guinea fowl. A few cases became uniformly pigmented and appeared to represent nonspecific patterns. The reciprocal grafts (guinea fowl wing buds grafted to the quail) became pigmented all over. We found evidence that the timing of melanocyte differentiation is controlled by cues in the feather papillae. Some cases developed a severe inflammatory response. The model which best accounts for these findings--and which can account for inconsistencies in previous reports--is the following. A prepattern is present in the feathers and this can control the differentiation of melanoblasts, even if they come from a different species. The local cues which constitute the prepattern are not positional values. In some chimeras melanoblasts fail to respond to the prepattern and so a nonspecific pattern of uniform pigmentation is produced.     

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.     

Endothelin signalling in the development of neural crest-derived melanocytes.

In both mice and humans, mutations in the genes encoding the endothelin B receptor and its ligand endothelin 3 lead to deficiencies in neural crest-derived melanocytes and enteric neurons. The discrete steps at which endothelins exert their functions in melanocyte development were examined in mouse neural crest cell cultures. Such cultures, kept in the presence of fetal calf serum, gave rise to cells expressing the early melanoblast marker Dct even in the absence of the phorbol ester tetradecanoyl phorbol acetate (TPA) or endothelins. However, these early Dct+ cells did not proliferate and pigmented cells never formed unless TPA or endothelins were added. In fact, endothelin 2 was as potent as TPA in promoting the generation of both Dct+ melanoblasts and pigmented cells, and endothelin 1 or endothelin 3 stimulated the generation of melanoblasts and of pigmented cells to an even greater extent. The inhibition of this stimulation by the selective endothelin B receptor antagonist BQ-788 (N-cis-2,6-dimethylpiperidinocarbonyl-L-alpha-methylleucyl-D -1-methoxycarbonyltryptophanyl-D-norleucine) suggested that the three endothelins all signal through the endothelin B receptor. This receptor was indeed expressed in Dct+ melanoblasts, in addition to cells lacking Dct expression. The results demonstrate that endothelins are potent stimulators of melanoblast proliferation and differentiation.     

Reversibility of phorbol diester-induced macrophage spreading

Phorbol 12-myristate 13-acetate and phorbol 12, 13-dibutyrate induced spreading of mouse macrophages with 50% effective concentrations of 3 nM and 35 nM, respectively. Macrophages treated with 100 or 1000 nM phorbol 12, 13- dibutyrate showed a time related decrease in spreading after washout. Spreading induced by 1, 10, or 100 nM phorbol 12-myristate 13-acetate was irreversible; however, washed phorbol 12,13-dibutyrate-treated cells respread after a second exposure to this compound. Washout of ³[H]phorbol diesters corroborated these observations in that 5% of ³H-phorbol 12-myristate 13-acetate and only 0.1% ³[H]phorbol, 12,13-dibutyrate remained associated with washed cells. Since phorbol 12-myristate 13 acetate is much more lipophilic than phorbol 12,13-dibutyrate, the reversibility of phorbol diester-induced macrophage spreading may depend upon the lipophilicity of the derivative utilized.Abbreviations DMEM Dulbecco's Minimal Essential Medium - PDA phorbol 12,13-diacetate - PDBu phorbol 12, 13-dibutyrate - PMA phorbol 12 myristate, 13 acetate - 4PDD phorbol 12, 13 didecanoate     

Analysis of phorbol ester receptors in phorbol ester unresponsive 3T3 cell variants

The phorbol ester tumor promoters induce multiple cellular responses in cell culture, including mitogenesis. We have analyzed 3 variants of mouse 3T3 cells mitogenically unresponsive to the phorbol esters for phorbol ester receptors. All resembled control 3T3 cells in their specific [³H]phorbol 12,13-dibutyrate binding. The variants thus appear to be altered at steps distal to receptor occupancy in the mitogenic response to the phorbol esters.     

Partial restriction in the developmental potential of late emigrating avian neural crest cells.

Trunk neural crest cells migrate along two major pathways: a ventral pathway through the somites whose cells form neuronal derivatives and dorsolateral pathway underneath the ectoderm whose cells become pigmented. In avian embryos, the latest emigrating neural crest cells move only along the dorsolateral pathway. To test whether late emigrating neural crest cells are more restricted in developmental potential than early migrating cells, cultures were prepared from the neural tubes of embryos at various stages of neural crest cell migration. "Early" and "middle" aged neural crest cells differentiated into many derivatives including pigmented cells, neurofilament-immunoreactive cells, and adrenergic cells. In contrast, "late" neural crest cells differentiated into pigment cells and neurofilament-immunoreactive cells, but not into adrenergic cells even after 10-14 days. To further challenge the developmental potential of early and late emigrating neural crest cells, they were transplanted into embryos during the early phases of neural crest cell migration, known to be permissive for adrenergic neuronal differentiation. The cells were labeled with the vital dye, DiI, and injected onto the ventral pathway at stages 14-17. Two and three days after injection, some early neural crest cells were found to express catecholamines, suggesting they were adrenergic neuroblasts. In contrast, DiI-labeled late neural crest cells never became catecholamine-positive. These results suggest that the late emigrating neural crest cell population has a more restricted developmental potential than the early migrating neural crest cell population.     

Stimulation of 86Rb+ and 32Pi movements in 3T3 cells by prostaglandins and phorbol esters

The potent tumor promoter tetradecanoyl phorbol acetate (TPA) induces early changes in ion movements analogous to those induced by prostaglandins E1 and F 2alpha. Among the earliest changes induced by TPA is a significant increase in 32Pi incorporation within 15 minutes incubation of TPA (10(-8)-10(-6) M) with post-confluent Swiss 3T3 mouse embryonic fibroblasts. Similarly, the active phorbol ester homolog 4-beta-OH phorbol didecanoate but not the inactive stereoisomeric 4-alpha-OH phorbol didecanoate stimulated 32Pi incorporation. Also, TPA at the above concentrations stimulated 86Rb+ influx shortly after administration. Both fluxes were ouabain-sensitive in accord with the idea that an early effect of TPA is to alter (Na+ + K+)-ATPase activity. Further, prostaglandin E1 (10(-7)-10(-6) M) and prostaglandin F 2alpha (3 X 10(-9)-10(-7) M) caused a similar stimulation of 86Rb+ and 32Pi uptake. The finding that water-soluble prostaglandin F 2alpha also exhibited stimulatory effects indicated that those hormone-induced responses are not mediated by solvent interactions. The similar responses of phorbol esters and prostaglandin derivatives suggests that phorbol esters and prostaglandin derivatives may act at common membrane sites. The finding that stimulatory effects were observed at discrete times in the logarithmic phase of growth suggests that the activation of membrane receptors may be cell-cycle dependent.     

Structural analogies between protein kinase C activators

Phorbol esters and diacylglycerols activate protein kinase C but specific structural parameters appear to be required for the enzyme activation. We have analyzed the conformation of potent and not potent diacylglycerols and phorbol esters. The orientation of the CH20H group at C3 of 1,2 diolein is remarkably similar to that of the same group at C-20 of 4 beta phorbol didecanoate and crucial for potency in activating the enzyme. Our data suggest that the new conformational approach here described could be used to rationally design specific inhibitors preventing the effects of tumor promoters and to predict the structure of potential tumor promoters.     

The lipophilicity of phorbol esters as a critical factor in determining the pattern of translocation of protein kinase C delta fused to green fluorescent protein

Our previous study showed differential subcellular localization of protein kinase C (PKC) delta by phorbol esters and related ligands, using a green fluorescent protein-tagged construct in living cells. Here we compared the abilities of a series of symmetrically substituted phorbol 12,13-diesters to translocate PKC delta. In vitro, the derivatives bound to PKC with similar potencies but differed in rate of equilibration. In vivo, the phorbol diesters with short, intermediate, and long chain fatty acids induced distinct patterns of translocation. Phorbol 12,13-dioctanoate and phorbol 12,13-nonanoate, the intermediate derivatives and most potent tumor promoters, showed patterns of translocation typical of phorbol 12-myristate 13-acetate, with plasma membrane and subsequent nuclear membrane translocation. The more hydrophilic compounds (phorbol 12,13-dibutyrate and phorbol 12,13-dihexanoate) induced a patchy distribution in the cytoplasm, more prominent nuclear membrane translocation, and little plasma membrane localization at all concentrations examined (100 nM to 10 microM). The highly lipophilic derivatives, phorbol 12,13-didecanoate and phorbol 12, 13-diundecanoate, at 1 microM caused either plasma membrane translocation only or no translocation at incubation times up to 60 min. Our results indicate that lipophilicity of phorbol esters is a critical factor contributing to differential PKC delta localization and thereby potentially to their different biological activities.     

Neurotrophin-3- and norepinephrine-mediated adrenergic differentiation and the inhibitory action of desipramine and cocaine

In the presence of neurotrophin-3 (NT-3), high-affinity norepinephrine (NE) uptake by quail neural crest cells was significantly increased as judged by in vitro colony assay of adrenergic differentiation. In the presence of the related neurotrophins nerve growth factor (NGF) or brain-derived neurotrophic (BDNF) factor, or of basic fibroblast growth factor (bFGF), there were no significant changes. When NE was added to the culture medium in addition to NT-3, more colonies contained dopamine-β-hydroxylase (DBH)-immunoreactive cells, an enzyme that is characteristic for adrenergic cells. The NE-mediated increase in the portion of colonies that contained DBH-immunoreactive cells was prevented by the tricyclic antidepressant desipramine (DMI) and by cocaine, two types of drug that block cellular transport of NE. To further examine whether NE acts via uptake, colony assays were performed in the presence and absence of adrenergic antagonists and agonists. These would be expected to mimic the DMI and NE effects, respectively, if the mechanism of action involved activation of adrenergic autoreceptors. Neither class of drug showed a detectable effect within a wide range of concentrations. Immunocytochemistry using antibodies against β1 and β2 adrenergic receptors further supported the notion that DMI action and β-receptor expression are not causally related. Ratio imaging was subsequently used in an attempt to elucidate the mechanism of NE action. Within a few minutes of addition of NE to the culture medium, there was an increase in intracellular free calcium in a subset of neural crest cells. Taken together, our data indicate that NT-3 is involved in the appearance of the NE transporter (NET) during embryonic development; internalized NE directly or indirectly increases adrenergic differentiation as measured by immunoreactivity of the adrenergic biosynthetic enzyme DBH; and norepinephrine uptake inhibitors have teratogenic potential. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 262–280, 1997.