Control of melanoblast differentiation in amphibia by alpha-melanocyte stimulating hormone, a serum melanization factor, and a melanization inhibiting factor

A ventrally localized melanization inhibiting factor (MIF) has been suggested to play an important role in the establishment of the dorsal-ventral pigment pattern in Xenopus laevis [Fukuzawa and Ide:Dev. Biol., 129:25-36, 1988]. To examine the possibility that melanoblast expression might be controlled by local putative MIF and melanogenic factors, the effects of alpha-melanocyte stimulating hormone (alpha-MSH), a serum melanization factor (SMF) from X. laevis or Rana pipiens, and MIF on the "outgrowth" and "melanization" of Xenopus neural crest cells were studied. Outgrowth represents the number of neural crest cells emigrating from cultured neural tubes, and melanization concerns the percentage of differentiated melanophores among the emigrated cells. MSH or SMF stimulate both outgrowth and melanization. The melanogenic effect of Xenopus serum in this system is more than twice that of Rana serum. The actions of MSH and Xenopus serum on melanization seem to be different: 1) Stronger melanization is induced by Xenopus serum than by MSH, and the onset of melanization occurs earlier with Xenopus serum; 2) MSH stimulates melanization only in the presence of added tyrosine; and 3) MSH causes young melanophores to assume a prominent state of melanophore dispersion during culture, while Xenopus serum (10%) had only a slight dispersing effect and not until day 3. A fraction of Xenopus serum presumably containing molecules of a smaller molecular weight (MW less than 30 kDa) than that of a pigment promoting factor reported in calf serum [Jerdan et al.: J. Cell Biol., 100:1493-1498, 1985] produces the same remarkable melanogenic effects as does intact serum. While this fraction stimulates outgrowth, another fraction presumably containing larger molecules (MW greater than 100 kDa) does not. MIF contained in Xenopus ventral skin conditioned medium (VCM) inhibits both outgrowth and melanization dose dependently. When VCM is used in combination with MSH, the stimulating effects of MSH on both outgrowth and melanization are completely inhibited. In contrast, the stimulatory effects of Xenopus serum are not completely inhibited when combined with VCM, although melanization is reduced to approximately 40% that of controls. MIF activity was also found to be present in ventral, but not in dorsal, skin conditioned media of R. pipiens when tested in the Xenopus neural crest system.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of ultraviolet light on melanocyte differentiation: studies with mouse neural crest cells and neural crest-derived cell lines

To evaluate the etiologic role of ultraviolet (UV) radiation in acquired dermal melanocytosis (ADM), we investigated the effects of UVA and UVB irradiation on the development and differentiation of melanocytes in primary cultures of mouse neural crest cells (NCC) by counting the numbers of cells positive for KIT (the receptor for stem cell factor) and for the L-3,4-dihydroxyphenylalanine (DOPA) oxidase reaction. No significant differences were found in the number of KIT- or DOPA-positive cells between the UV-irradiated cultures and the non-irradiated cultures. We then examined the effects of UV light on KIT-positive cell lines derived from mouse NCC cultures. Irradiation with UVA but not with UVB inhibited the tyrosinase activity in a tyrosinase-positive cell line (NCCmelan5). Tyrosinase activity in the cells was markedly enhanced by treatment with alpha-melanocyte-stimulating hormone (alpha-MSH), but that stimulation was inhibited by UVA or by UVB irradiation. Irradiation with UVA or UVB did not induce tyrosinase activity in a tyrosinase-negative cell line (NCCmelb4). Levels of KIT expression in NCCmelan5 cells and in NCCmelb4 cells were significantly decreased after UV irradiation. Phosphorylation levels of extracellular signal-regulated kinase 1/2 in cells stimulated with stem cell factor were also diminished after UV irradiation. These results suggest that UV irradiation does not stimulate but rather suppresses mouse NCC. Thus if UV irradiation is a causative factor for ADM lesions, it would not act directly on dermal melanocytes but may act in indirect manners, for instance, via the overproduction of melanogenic cytokines such as alpha-MSH and/or endothelin-1.     

Induction of melanogenesis by tetradecanoylphorbol-13 acetate and endothelin 3 in embryonic avian peripheral nerve cultures

In this study, we have analyzed the melanogenic potential of Schwann cells using in vitro cell cultures of embryonic quail peripheral nerves. It is shown that in Schwann cells, two factors, 12-O-tetradecanoylphorbol-13 acetate (TPA) and endothelin 3, trigger a differentiation pathway toward melanocytes, and that Steel factor has no effect on these cells unless treated simultaneously with TPA. In these cultures, TPA induces the expression of c-kit, whereas Steel factor enhances the development of melanocytes. In the assay system we employed, neither neuronal nor catecholaminergic phenotypes were obtained, regardless of various combinations of related factors added to the culture medium. These data support our previous observations indicating the existence of bipotent progenitors that are capable of differentiating into Schwann cells or into melanocytes, and the regulatory role of endothelin 3 on those precursors, as revealed by the clonal culture of neural crest cells.     

The sympathoadrenal lineage in avian embryos. II. Effects of glucocorticoids on cultured neural crest cells

The neural crest-derived precursors of the sympathoadrenal lineage depend on environmental cues to differentiate as sympathetic neurons and pheochromocytes. We have used the monoclonal antibody A2B5 as a marker for neuronal differentiation and antisera against catecholamine synthesis enzymes to investigate the differentiation of catecholaminergic cells in cultures of quail neural crest cells. Cells corresponding phenotypically to sympathetic neurons and pheochromocytes can be identified in neural crest cell cultures after 5-6 days in vitro. Expression of the A2B5 antigen precedes expression of immunocytochemically detectable levels of tyrosine hydroxylase in cultured neural crest cells. Glucocorticoid treatment decreases the proportion of TH+ neural crest cells that express neuronal traits. We conclude that environmental cues normally encountered by sympathoadrenal precursors in vivo can influence the differentiation of a subpopulation of cultured neural crest cells in the sympathoadrenal lineage.     

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.     

From the Crest to the Periphery: Control of Pigment Cell Migration and Lineage Segregation

Pigment cells are one of many cell types derived from the neural crest. This review focuses on the mechanisms that control the timing and pathways of migration of pigment cells into the epidermis and determinants that control the differentiation of pigment cells. Several factors may control the timing and pattern of pigment cell migration in the dorsolateral space including the loss of inhibitory molecules in the pathway, the appearance of chemotactic molecules emanating from the dispersing dermatome, and the differentiation of pigment cells, which may be the only neural crest derivative capable of utilizing the substratum found in the dorsolateral path Control of pigment cell differentiation remains controversial. A working model presented in this review suggests that multipotent neural crest cells that disperse ventrally upon separation from the neural tube preserve neurogenic ability and lose melanogenic ability, whereas those cells that are arrested at the entrance to the dorsolateral path lose neurogenic ability so that the population becomes primarily melanogenic. During the time that the latter population is arrested in migration it is speculated that the neural crest cells are exposed to an environment comprised of specific extracellular matrix molecules and/or growth factors that enhance pigment cell differentiation.     

Effects of Ultraviolet Light on Melanocyte Differentiation: Studies with Mouse Neural Crest Cells and Neural Crest‐derived Cell Lines

To evaluate the etiologic role of ultraviolet (UV) radiation in acquired dermal melanocytosis (ADM), we investigated the effects of UVA and UVB irradiation on the development and differentiation of melanocytes in primary cultures of mouse neural crest cells (NCC) by counting the numbers of cells positive for KIT (the receptor for stem cell factor) and for the L ‐3,4‐dihydroxyphenylalanine (DOPA) oxidase reaction. No significant differences were found in the number of KIT‐ or DOPA‐positive cells between the UV‐irradiated cultures and the non‐irradiated cultures. We then examined the effects of UV light on KIT‐positive cell lines derived from mouse NCC cultures. Irradiation with UVA but not with UVB inhibited the tyrosinase activity in a tyrosinase‐positive cell line (NCCmelan5). Tyrosinase activity in the cells was markedly enhanced by treatment with α‐melanocyte‐stimulating hormone (α‐MSH), but that stimulation was inhibited by UVA or by UVB irradiation. Irradiation with UVA or UVB did not induce tyrosinase activity in a tyrosinase‐negative cell line (NCCmelb4). Levels of KIT expression in NCCmelan5 cells and in NCCmelb4 cells were significantly decreased after UV irradiation. Phosphorylation levels of extracellular signal‐regulated kinase 1/2 in cells stimulated with stem cell factor were also diminished after UV irradiation. These results suggest that UV irradiation does not stimulate but rather suppresses mouse NCC. Thus if UV irradiation is a causative factor for ADM lesions, it would not act directly on dermal melanocytes but may act in indirect manners, for instance, via the overproduction of melanogenic cytokines such as α‐MSH and/or endothelin‐1.     

Induction of Melanogenesis by Tetradecanoylphorbol‐13 Acetate and Endothelin 3 in Embryonic Avian Peripheral Nerve Cultures

In this study, we have analyzed the melanogenic potential of Schwann cells using in vitro cell cultures of embryonic quail peripheral nerves. It is shown that in Schwann cells, two factors, 12‐O‐tetradecanoylphorbol‐13 acetate (TPA) and endothelin 3, trigger a differentiation pathway toward melanocytes, and that Steel factor has no effect on these cells unless treated simultaneously with TPA. In these cultures, TPA induces the expression of c‐kit, whereas Steel factor enhances the development of melanocytes. In the assay system we employed, neither neuronal nor catecholaminergic phenotypes were obtained, regardless of various combinations of related factors added to the culture medium. These data support our previous observations indicating the existence of bipotent progenitors that are capable of differentiating into Schwann cells or into melanocytes, and the regulatory role of endothelin 3 on those precursors, as revealed by the clonal culture of neural crest cells.     

Dibutyrylic cyclic AMP and theophylline inhibit proliferation of accessory cells in primary cultures of adrenomedullary cells

Summary Studies on isolated adrenal chromaffin cells in primary cultures may be seriously hampered by the presence of non-chromaffin, mainly fibroblast-like cells, which always occur in dissociates of adrenal medullary tissue and often outnumber the chromaffin cells by the end of the first week of culture, when no measures are taken to control their proliferation. The present study offers a new means to inhibit effectively the proliferation of these accessory cells by treating the cultures with dibutyrylic cyclic AMP (dbcAMP, 0.1 or 0.01 mM) and equimolar amounts of the phosphodiesterase inhibitor theophylline. With this treatment cultures of young rat adrenal chromaffin cells remain virtually free of accessory cells for two weeks of culture. Cultures of bovine adrenomedullary cells retain their initial amounts of non-chromaffin cells, which largely depends upon whether the primary cell suspensions have undergone differential plating prior to seeding. Suppression of accessory cell proliferation with dbcAMP and theophylline is partly due to maintaining differentiation of cortical cells, which otherwise dedifferentiate into rapidly dividing fibroblast-like elements. However, a more direct action of dbcAMP on accessory cells in terms of growth control is also conceivable. DbcAMP and theophylline in the doses applied do not impair the viability, ultrastructure and catecholamine-storing capacity of cultured chromaffin cells.     

Control of Melanoblast Differentiation in Amphibia by α-Melanocyte Stimulating Hormone,a Serum Melanization Factor,and a Melanization Inhibiting Factor

A ventrally localized melanization inhibiting factor (MIF) has been suggested to play an important role in the establishment of the dorsal-ventral pigment pattern in Xenopus laevis [Fukuzawa and Ide: Dev. Biol., 129:25–36, 1988]. To examine the possibility that melanoblast expression might be controlled by local putative MIF and melanogenic factors, the effects of α-melanocyte stimulating hormone (α-MSH), a serum melanization factor (SMF) from X. laevis or Rana pipiens, and MIF on the “outgrowth” and “melanization” of Xenopus neural crest cells were studied. Outgrowth represents the number of neural crest cells emigrating from cultured neural tubes, and melanization concerns the percentage of differentiated melanophores among the emigrated cells. MSH or SMF stimulate both outgrowth and melanization. The melanogenic effect of Xenopus serum in this system is more than twice that of Rana serum. The actions of MSH and Xenopus serum on melanization seem to be different: 1) Stronger melanization is induced by Xenopus serum than by MSH, and the onset of melanization occurs earlier with Xenopus serum; 2) MSH stimulates melanization only in the presence of added tyrosine; and 3) MSH causes young melanophores to assume a prominent state of melanophore dispersion during culture, while Xenopus serum (10%) had only a slight dispersing effect and not until day 3. A fraction of Xenopus serum presumably containing molecules of a smaller molecular weight (MW <30 kDa) than that of a pigment promoting factor reported in calf serum [Jerdan et al.: J. Cell Biol., 100:1493–1498, 1985] produces the same remarkable melanogenic effects as does intact serum. While this fraction stimulates outgrowth, another fraction presumably containing larger molecules (MW > 100 kDa) does not. MIF contained in Xenopus ventral skin conditioned medium (VCM) inhibits both outgrowth and melanization dose dependently. When VCM is used in combination with MSH, the stimulating effects of MSH on both outgrowth and melanization are completely inhibited. In contrast, the stimulatory effects of Xenopus serum are not completely inhibited when combined with VCM, although melanization is reduced to approximately 40% that of controls. MIF activity was also found to be present in ventral, but not in dorsal, skin conditioned media of R. pipiens when tested in the Xenopus neural crest system. We suggest that ventrally localized MIF plays an important role in amphibian pigment pattern formation and that the interacting effects of MIF and melanogenic factors influence melanoblast differentiation, migration, and/or proliferation of neural crest cells to effect the expression of pigmentary patterns.     

Cultured quail neural crest cells attain competence for terminal differentiation into melanocytes before competence to terminal differentiation into adrenergic neurons

At the onset of migration the quail neural crest contains pluripotent progenitor cells that give rise to both melanocytes and adrenergic neurons as well as progenitor cells that are already committed to the melanogenic or the neuronal pathway. In this paper we show that melanogenic progenitors attain the competence for terminal differentiation prior to adrenergic progenitors. The adrenergic phenotype was only expressed when the crest cells were allowed to proliferate in vitro for at least 3 days. Differentiation into melanocytes, however, occurred even when proliferation was blocked with cytosine arabinoside immediately after explantation of the neural tube.     

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.     

Tumor-Promoting Phorbol Esters Promote Melanogenesis and Prevent Expression of the Adrenergic Phenotype in Quail Neural Crest Cells

Tumor-promoting phorbol esters were used to manipulate the in vitro development of neural crest cells. When plated at clonal density in secondary culture, quail neural crest cells from the trunk region gave rise to three types of colonies, pigmented, unpigmented, and mixed. Pigmented colonies consisted exclusively of melanocytes; up to 50% of the unpigmented and mixed colonies contained adrenergic nerve cells which could be identified by a catecholamine-specific histofluorescence method. Addition of potent tumor promoters to the culture medium shortened the doubling time of neural crest cells and altered their morphologic appearance. It also delayed the onset of pigmentation, prevented the expression of the adrenergic phenotype, reduced the number of unpigmented and mixed colonies, and increased the number of pigmented colonies, most likely by directing progenitor cells preferentially to the melanogenic pathway. There was a clear correlation between the ability of phorbol esters to promote skin tumors in mice and their ability to interfere with the in vitro development of quail neural crest cells. The potent promoters 12–0–tetradecanoyl phorbol 13–acetate (TPA) and phorbol 12,13–didecanoate (PDD) were most effective, phorbol 12,13–diacetate (PDA) was considerably less effective, the nonpromoting analogues 4–0–methyl 12–0–tetradecanoyl phorbol 13–acetate (4–0–Me-TPA) and 4α-phorbol 12,13–didecanoate (4α-PDD) and the parent alcohol phorbol (PHR) had little or no effect.     

Extracellular matrix from normal but not Steel mutant mice enhances melanogenesis in cultured mouse neural crest cells

The Steel mutation is a non-cell-autonomous defect in mice that affects the development of several stem cell populations, including germ cells, hematopoietic cells, and neural crest-derived pigment cells. To characterize the environmental lesion caused by the Steel mutation, we have compared the ability of normal and mutant extracellular matrix material to support the differentiation of normal mouse neural crest cells in vitro. Extracellular matrix deposited by cultured skin cells isolated from normal fetuses enhanced melanogenesis by crest cells over that observed on plastic substrata. In contrast, matrix material produced by Steel-Dickie (Sld) fetal skin cells failed to enhance melanogenesis. Adrenergic differentiation by neural crest-derived cells was promoted equally by both normal and mutant extracellular matrix compared to control substrata. We conclude that the environmental defect in mutant embryos selectively affects a melanogenic subpopulation of neural crest cells and resides, at least in part, in the extracellular matrix.     

Differentiation of neural crest cells of Xenopus laevis in clonal culture

Clonal cultures were performed with the use of neural crest cells and their derivatives, chromatophores, from Xenopus laevis in order to elucidate the state of commitment in early embryogenesis. Neural crest cells that outgrew from neural tube explants were isolated and plated at clonal density. Cloned neural crest cells differentiated and gave rise to colonies that consisted of 1) only melanophores, 2) only xanthophores, or 3) melanophores and xanthophores. Xanthophores and iridophores, which differentiated in vitro, were also isolated and cloned. Cloned xanthophores proliferated in a stable fashion and did not lose their properties. On the other hand, cloned iridophores converted into melanophores as they proliferated. These results suggest that there is heterogeneity in the state of commitment of neural crest cells immediately after migration with regard to chromatophore differentiation and that iridophore determination is relatively labile (at least in vitro), whereas melanophore and xanthophore phenotypes are stable.     

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.     

Expression of c-kit protooncogene is stimulated by cAMP in differentiated F9 mouse teratocarcinoma cells.

Protooncogene c-kit, a transmembrane tyrosine kinase receptor, was recently shown to map to the dominant white spotting locus (W) of the mouse. W mutations affect melanogenesis, gametogenesis, and hematopoiesis during development and in adult life. In order to determine the regulation of the c-kit gene in cell differentiation, we investigated its expression during the differentiation of F9 cells. Undifferentiated F9 cells and F9 cells treated with retinoic acid (RA) alone or dbcAMP alone showed little expression of c-kit mRNA if any. The subsequent addition of dbcAMP to F9 cells treated with RA markedly increased the expression of c-kit mRNA. Furthermore, the effect of dbcAMP on c-kit expression is reversible. In differentiated cells treated with RA, c-kit gene expression is induced by agents such as forskolin or theophylline, which are known to elevate cellular cAMP level. These results indicate that the expression of the c-kit gene is regulated by the level of intracellular cAMP in differentiated F9 cells induced by RA.     

Expression of melanocyte stimulating hormone receptors correlates with mammalian pigmentation, and can be modulated by interferons

The relationship between melanogenesis and the expression of melanocyte stimulating hormone (MSH) receptors on the surface of melanocytes was examined using sublines generated from the melanotic JB/MS melanoma. JB/MS cells were propagated in long term culture to allow for phenotypic drift in their characteristics of differentiation, and then were cloned; the cloned cells ranged from well differentiated and pigmented to undifferentiated and amelanotic. Spontaneous and MSH-induced melanogenesis in these different lines was measured and correlated with the number of MSH receptors expressed. After 6 months of in vitro culture, the ability of the cells to respond to MSH was significantly reduced, as were the number of MSH receptors expressed; the cells had reduced pigmentation and were relatively undifferentiated histologically. Subsequently, clonally-derived pigmented cells were found to have numbers of surface MSH receptors (approximately 60,000 per cell) and levels of melanogenic activity similar to the original JB/MS cell line. However, an amelanotic clone had an even more dramatically reduced level of pigmentation which correlated with a further decrease in the expression of MSH receptors (less than 1,000 per cell) and the production of a potent melanogenic inhibitor. We also examined the responses of these various sublines to alpha, beta, and gamma-interferons and found significant heterogeneity in their abilities to respond to these cytokines. This study clearly shows that there is a direct correlation between melanogenesis and the expression of MSH receptors on the surface of melanocytes, and that melanogenic inhibitors may be critically involved in the regulation of mammalian pigmentation.