Studies of pigment transfer between Xenopus laevis melanophores and fibroblasts in vitro and in vivo1

Frog melanophores rapidly change colour by dispersion or aggregation of melanosomes. A long‐term colour change exists where melanosomes are released from melanophores and transferred to surrounding skin cells. No in vitro model for pigment transfer exists for lower vertebrates. Frog melanophores of different morphology exist both in epidermis where keratinocytes are present and in dermis where fibroblasts dominate. We have examined whether release and transfer of melanosomes can be studied in a melanophore‐fibroblast co‐culture, as no frog keratinocyte cell line exists. Xenopus laevis melanophores are normally cultured in conditioned medium from fibroblasts and fibroblast‐derived factors may be important for melanophore morphology. Melanin was exocytosed as membrane‐enclosed melanosomes in a process that was upregulated by α‐melanocyte‐stimulating hormone (α‐MSH), and melanosomes where taken up by fibroblasts. Melanosome membrane‐proteins seemed to be of importance, as the cluster‐like uptake pattern of pigment granules was distinct from that of latex beads. In vivo results confirmed the ability of dermal fibroblasts to engulf melanosomes. Our results show that cultured frog melanophores can not only be used for studies of rapid colour change, but also as a model system for long‐term colour changes and for studies of factors that affect pigmentation.     

Proliferation in vitro of melanophores from Xenopus laevis

Melanophores of wild-type and periodic albino mutants of Xenopus laevis were successfully cultured in vitro. They proliferated in the presence of alpha-melanocyte-stimulating hormone (alpha-MSH or cyclic adenosine monophosphate (cAMP) at a doubling time of 8-10 days. These proliferating melanophores retained their phenotypes, ability to synthesize melanin, and melanin-dispersing response to MSH stimulation. Neither depigmentation nor selective cell death of periodic albino melanophores was observed for at least 4 months during the cultivation.     

Nitric oxide modulates intracellular translocation of pigment organelles in Xenopus laevis melanophores

Pigment organelles in Xenopus laevis melanophores are used by the animal to change skin color, and they provide a good model for studying intracellular organelle transport. Movement of organelles and vesicles along the cytoskeleton is essential for many processes, such as axonal transport, endocytosis, and intercompartmental trafficking. Nitric oxide (NO) is a signaling molecule that plays a role in, among other things, relaxation of blood vessels, sperm motility, and polymerization of actin. Our study focused on the effect NO exerts on cytoskeleton-mediated transport, which has previously received little attention. We found that an inhibitor of NO synthesis, N-nitro-L-arginine methyl ester (L-NAME), reduced the melatonin-induced aggregation of the pigment organelles, melanosomes. Preaggregated melanosomes dispersed after treatment with L-NAME but not after exposure to the inactive stereoisomer (D-NAME) or the substrate for NO synthesis (L-arginine). Signal transduction by NO can be mediated through the activation of soluble guanylate cyclase (sGC), which leads to increased production of cGMP and activation of cGMP-dependent kinases (PKG). We found that both the sGC inhibitor 1H-(1,2,4) oxadiazolo(4,3-a)quinoxalin-1-one (ODQ) and the cGMP analogue 8-bromoguanosine 3':5'-cyclic monophosphate (8-Br-cGMP) reduced melanosome aggregation, whereas the PKG inhibitor KT582 did not. Our results demonstrate that melanosome aggregation depends on synthesis of NO, and NO deprivation causes dispersion. It seems, thus, as if NO and cGMP are essential and can regulate melanosome translocation.     

Protein kinase C activation antagonizes melatonin-induced pigment aggregation in Xenopus laevis melanophores

The pineal hormone, melatonin (5-methoxy N-acetyltryptamine) induces a rapid aggregation of melanin-containing pigment granules in isolated melanophores of Xenopus laevis. Treatment of melanophores with activators of protein kinase C (PKC), including phorbol esters, mezerein and a synthetic diacylglycerol, did not affect pigment granule distribution but did prevent and reverse melatonin-induced pigment aggregation. This effect was blocked by an inhibitor of PKC, Ro 31-8220. The inhibitory effect was not a direct effect on melatonin receptors, per se, as the slow aggregation induced by a high concentration of an inhibitor of cyclic AMP-dependent protein kinase (PKA), adenosine 3',5'-cyclic monophosphothioate, Rp-diastereomer (Rp-cAMPS), was also reversed by PKC activation. Presumably activation of PKC, like PKA activation, stimulates the intracellular machinery involved in the centrifugal translocation of pigment granules along microtubules. alpha-Melanocyte stimulating hormone (alpha-MSH), like PKC activators, overcame melatonin-induced aggregation but this response was not blocked by the PKC inhibitor, Ro 31-8220. This data indicates that centrifugal translocation (dispersion) of pigment granules in Xenopus melanophores can be triggered by activation of either PKA, as occurs after alpha-MSH treatment, or PKC. The very slow aggregation in response to inhibition of PKA with high concentrations of Rp-cAMPS, suggests that the rapid aggregation in response to melatonin may involve multiple intracellular signals in addition to the documented Gi-mediated inhibition of adenylate cyclase.     

Studies on cellular adhesion of Xenopus laevis melanophores: pigment pattern formation and alteration in vivo by endogenous galactoside-binding lectin or its sugar hapten inhibitor

We have studied the development of Xenopus laevis tail melanophores and the effects on these cells on confrontation with endogenous X. laevis galactoside-binding lectin or its sugar hapten inhibitor thiodigalactoside (TDG). An initial population of unpigmented cells differentiates into melanophores on the dorsal surface of the neural tube, and on the dorsal and ventral apices of the myotomes, forming the larval pattern. Melanophores secondarily populate the flank, forming a spaced arrangement which is later transformed into a dorsal and ventral strip. A technique has been developed for confrontation of premigratory neural crest with purified lectin or TDG. These molecules impact on tail melanophores. With lectin treatment melanophore numbers decrease, and cell morphologies and arrangements change. TDG treatment, however, primarily affects pigment cell morphology. These results suggest that both galactoside-bearing receptors for this lectin and the lectin itself can affect melanophores in this species of frog.     

The effect of cytochalasin B on pigment dispersion and aggregation in perfused Xenopus laevis tailfin melanophores

A perfusion technique is described for the study of melanosome response in ventral tailfin melanophores of Xenopus laevis tadpoles. The melanosomes remain aggregated (punctate melanophores) in Ringer's. Theophylline (15 mM) and caffeine (30 mM) cause a reversible dispersion (stellate melanophores) of melanosomes which is partly blocked by cytochalasin B (10 μg/ml). When added with theophylline or caffeine to stellate cells, cytochalasin B causes a disrupted distribution of pigment granules, characterized by a melanosome free central region. C-AMP (20 mM) and dibutyryl c-AMP (1 mM) cause a reversible dispersion of melanosomes which is partly inhibited by cytochalasin. When cytochalasin plus a nucleotide are added to stellate cells, some show the disrupted distribution of melanosomes. Colchicine (5 mM) causes irreversible, while griseofulvin (0.2 mM) causes a slight, but reversible dispersion of melanosomes, and cytochalasin has little effect on these reactions. Perfused tailfin melanophores remain capable of responding to reversible reagents for at least 12 hours and are unresponsive to changes in illumination.     

Identification of microtubule-organizing centers in interphase melanophores of Xenopus laevis larvae in vivo.

The morphological characteristics of microtubule-organizing centers (MTOCs) in dermal interphase melanophores of Xenopus laevis larvae in vivo at 51-53 stages of development has been studied using immunostained semi-thick sections by fluorescent microscopy combined with computer image analysis. Computer image analysis of melanophores with aggregated and dispersed pigment granules, stained with the antibodies against the centrosome-specific component (CTR210) and tubulin, has revealed the presence of one main focus of microtubule convergence in the cell body, which coincides with the localization of the centrosome-specific antigen. An electron microscopy of those melanophores has shown that aggregation or dispersion of melanosomes is accompanied by changes in the morphological arrangement of the MTOC/centrosome. The centrosome in melanophores with dispersed pigment exhibits a conventional organization, and their melanosomes are situated in an immediate vicinity of the centrioles. In melanophores with aggregated pigment, MTOC is characterized by a three-zonal organization: the centrosome with centrioles, the centrosphere, and an outlying radial arrangement of microtubules and their associated inclusions. The centrosome in interphase melanophores is presumed to contain a pair of centrioles or numerous centrioles. Because of an inability of detecting additional MTOCs, it has been considered that an active MTOC in interphase melanophores of X. laevis is the centrosome. We assume that remaining intact microtubules in the cytoplasmic processes of mitotic melanophores (Rubina et al., 1999) derive either from the aster or the centrosome active at the interphase.     

Studies on cellular adhesion of Xenopus laevis melanophores: modulation of cell-cell and cell-substratum adhesion in vitro by endogenous Xenopus galactoside-binding lectin

We have investigated cell-cell and cell-substratum adhesion of Xenopus laevis neural crest cells at various stages of melanophore differentiation. Single-cell suspensions were obtained by trypsinization and aggregated in a cell-cell adhesion assay. Unpigmented cells did not adhere while the rate of adhesion of melanophores correlated with the degree of melanization. Melanophore cell-cell adhesion decreased significantly in the presence of beta-galactosidase, which suggests that cell-surface galactose is involved. Beta-galactoside-binding lectin has been isolated and purified from embryos at the stage of neural crest migration. When added to aggregating cells smaller, looser clusters formed compared to controls. When lectin was added to cells in stationary culture to test cell-substratum adhesion, melanophores spread more smoothly and formed more regular spacing patterns. These results suggest that this lectin can modulate receptors used in cell-cell and cell-substratum adhesion of melanophores.     

Microtubule-organizing centers in the mitotic melanophores of Xenopus laevis larvae in vivo: ultrastructural study

Mitotic melanophores of Xenopus laevis larvae at 51-53 stages of development were morphologically studied using light and electron microscopy, with special reference to their microtubule-organizing centers. These melanophores represented a highly branched cell shape in mitosis, each cell process is distributed with melanosomes without exhibiting any responsiveness to hormonal (melatonin) stimulation, and upon completion of mitosis, recovered the ability to translocate these granules in response to such a stimulus. At the metaphase, these cells contained bipolar or multipolar spindles, whose poles were composed of three zones: the centrosome with centrioles; the centrosphere; and an outlying radial arrangement of microtubules and their associated inclusions. In these mitotic melanophores, a number of microtubules are distributed within the radially stretching cell processes, whereas an abundance of microtubules reside in the spindles. Possible origins of the microtubules observed in these cytoplasmic processes are discussed in relation to the loss of the ability of pigment translocation during mitosis.     

Efficient, long-term transgene expression in Xenopus laevis dermal melanophores

Xenopus laevis dermal melanophores provide an excellent model system for the investigation of complex cellular processes. Specifically, the expression of exogenous genes in Xenopus melanophores is the basis of recombinant bioassays for the study of receptor-ligand interactions. However, due to their slow rate of cell division and to the relatively low efficiency of current transfection protocols, long-term expression of exogenous genes and the generation of stable melanophore cell lines remains problematic. In this report we demonstrate the efficient, long-term expression of two exogenous proteins, the enhanced green fluorescent protein (EGFP) and the human CD4 (hCD4) cell surface receptor, following stable introduction into Xenopus melanophores via an HIV-1 based vector. Transduction of melanophores with the EGFP expression vector resulted in up to 80% EGFP+ cells. After 1 year in continuous culture in the absence of antibiotic selection, more than 60% of the cells remained EGFP+. Furthermore, we demonstrate the expression of hCD4 melanophores for over 9 months in continuous culture in the absence of antibiotic selection. Our results indicate that lentivirus vectors provide an efficient means of introducing genetic information into Xenopus melanophores, resulting in sustained levels of gene expression. The significance of this gene transfer system for the study of cellular signal transduction pathways is discussed.     

Effect of colcemid on the centrosome and microtubules in dermal melanophores of Xenopus laevis larvae in vivo.

An electron microscopy study showed that in melanophores with dispersed and aggregated pigment the sensitivity of the centrosome and the stability of microtubules were different and depended on the colcemid concentration. The structure of the centrosome didn't change upon exposure to colcemid in dispersed melanophores. In aggregated melanophores, on exposure to 10(-6) M colcemid, the centrosome retained its structure; colcemid at 10(-5)-10(-3) M caused a dramatic collapse of the centrosome. Treatment of aggregated melanophores with colcemid resulted in the complete disassembly of the microtubules; though microtubules in dispersed melanophores appear to be colcemid resistant. Light microscopy studies indicated that in Xenopus melanophores with aggregated or dispersed pigment melanosomes didn't change their location after exposure to 10(-3)-10(-6) M colcemid. Subsequent incubation in colcemid-free medium revealed that the cells retained their ability to translocate melanosomes in response to hormone stimulation. Electron microscopy data revealed the inactivation of the centrosome as MTOC (microtubule-organizing center) in dispersed melanophores with melatonin substituted for MSH in the presence of colcemid. In contrast, with melanocyte-stimulating hormone (MSH) substituted for melatonin, we observed the activation of the centrosome in aggregated cells. We showed that in aggregated melanophores pigment movement proceeded in the complete absence of microtubules, suggesting the involvement of a microtubule-independent component in the hormone-induced melanosome dispersion. However, we observed abnormal aggregation along colcemid-resistent microtubules in dispersed melanophores, suggesting the involvement of not only stable but also labile microtubules in the centripetal movement of melanosomes. The results raise the intriguing questions about the mechanism of the hormone and colcemid action on the centrosome structure and microtubule network in melanophores with dispersed and aggregated pigment.     

A role for spectrin in dynactin-dependent melanosome transport in Xenopus laevis melanophores

The bi-directional movement of pigment granules in frog melanophores involves the microtubule-based motors cytoplasmic dynein, which is responsible for aggregation, and kinesin II and myosin V, which are required for dispersion of pigment. It was recently shown that dynactin acts as a link between dynein and kinesin II and melanosomes, but it is not fully understood how this is regulated and if more proteins are involved. Here, we suggest that spectrin, which is known to be associated with Golgi vesicles as well as synaptic vesicles in a number of cells, is of importance for melanosome movements in Xenopus laevis melanophores. Large amounts of spectrin were found on melanosomes isolated from both aggregated and dispersed melanophores. Spectrin and two components of the oligomeric dynactin complex, p150(glued) and Arp1/centractin, co-localized with melanosomes during aggregation and dispersion, and the proteins were found to interact as determined by co-immunoprecipitation. Spectrin has been suggested as an important link between cargoes and motor proteins in other cell types, and our new data indicate that spectrin has a role in the specialized melanosome transport processes in frog melanophores, in addition to a more general vesicle transport.     

Morphological and physiological aspects of melanophores in primary culture from tadpoles of Xenopus laevis

Summary Melanophores from tadpoles of Xenopus laevis (Daudin) were isolated by digestion of tail fins with acetyltrypsin and collagenase and maintained in primary culture for 6 weeks up to 3 months. Within 36 to 72 h the melanophores develop one to eight dendritic processes per cell; secondary and tertiary branchings of the processes were frequently observed. The melanophores in primary culture disperse under the influence of -MSH or cyclic AMP; upon rinsing out these substances the cells aggregate. In darkness, about 40 % of the cells disperse their pigment, whereas under illumination the pigment of the melanophores aggregates. To date, attempts to initiate cell division in melanophores have not been successful.     

Effect of cyclic AMP and cytochalasin B on tissue cultured melanophores of Xenopus laevis

The effects of cytochalasin B or low concentrations of adenosine 3′,5′-monophosphate (cyclic AMP) were tested on melanophores in hanging drop preparations of neural fold explants from Xenopus laevis embryos in Barths' solution. After one week in culture, the melanophores were punctate in this medium. Cyclic AMP at 5 mM consistently caused reversible morphological transformation of these cells to the stellate state, whether they were situated within an epithelial outgrowth or isolated on the surface of the coverglass. Only the isolated melanophores consistently responded to 1 mM cyclic AMP. Cytochalasin B at 1–10 μg/ml caused aggregation of melanin granules in stellate cells, but left long, narrow cell branches containing some melanosomes. Its effect was at least partially reversible and appeared to be dose dependent. At 1% concentration, dimethyl sulfoxide caused melanin dispersion.     

Reiterated transfer RNA genes of Xenopus laevis

The proportion of the Xenopus laevis genome complementary to “7 S” RNA, unfractionated transfer RNA and some selected aminoacyl-tRNAs, and the sequence complexity of these RNA species, have been determined by following the kinetics of RNA-DNA hybridization on filters under conditions of RNA excess at optimum rate temperature. For hybridization of aminoacyl-labelled tRNAs, conditions for optimum aminoacylation were first determined and, where necessary, aminoacyl-tRNAs were treated with nitrous acid to prevent discharge during annealing. Neither the extent nor rate of hybridization was affected by this treatment.“7 S” RNA, coded for by 580 genes per haploid complement of chromosomes, reacts like a single family of nucleotide sequences, whereas about 43 basic tRNA sequences are coded for by at least 7800 genes. If hybrids are not treated with RNase A, the apparent tDNA redundancy is some 23% greater but no more nucleotide sequences are detectable. Taken together, the results suggest that each tRNA sequence is, on average, 200-fold reiterated.The reiteration varies, however, for different aminoacyl-tRNAs. Thus, hybridization resolves only one valyl-tRNA which is coded for by 240 genes, but at least four leucyl-tRNA sequences can be distinguished by hybridization, each of which is on average 90-fold reiterated. Reiteration also varies for the two methionyl-tRNAs detectable both by hybridization and by reversed phase chromatography: tRNA₁^Met and tRNA₂^Met are 310- and 170-fold reiterated, respectively, and each is kinetically homogeneous. These saturation values are almost exactly additive and are not influenced by the presence of other tRNA species. Thus the results suggest that Xenopus tRNAs are no more heterogeneous than would be predicted by the genetic code, despite the high but variable multiplicity of tRNA cistrons.     

Comparison of in vivo and in vitro ribosomal RNA synthesis in nucleolar mutants of Xenopus laevis

Cells of embryos carrying a lethal nucleolar mutation have been maintained in vitro for extended periods of time. Normally these mutants live only 9 to 12 days after fertilization but their cells in culture will survive for more than 3 months. The extent of ribosomal RNA (rRNA) synthesis was determined in primary cultures prepared from normal embryos and nucleolar mutants having different numbers of ribosomal RNA genes. We found that the accumulation of radioactivity into rRNA for normal and mutant embryos was similar in vivo and in vitro. In primary cultures of normal embryos which have two nucleoli per cell and mutant embryos which have only one nucleolus per cell, the incorporation of radioactivity into rRNA was similar even though the normal cells have twice as many rRNA genes. Thus the mechanism which regulates dosage compensation of the rRNA genes operates both in vivo and in vitro.     

Studies on Maturation in Xenopus laevis Oocytes

The following results were obtained: 1) The oxygen comsumption of progesterone-stimulated X. laevis oocytes increases at the time of germinal vesicle (GV) breakdown. 2) Continous treatment with 1 mM KCN, 1 mM and 0.1 mM DNP completely inhibits GV breakdown. 3) Pretreatment experiments with KCN, DNP and cycloheximide show that binding of progesterone to the plasma membrane and the specific hormone receptor requires neither energy, nor protein synthesis. 4) 1 h pulses of DNP (1mM) or cycloheximide (50 mug/ml) were applied, at various time intervals after progesterone treatment: early pulses strongly delay, but do not prevent GV breakdown; late pulses just before GV breakdown induce a number of cytological abnormalities, which ultimately lead to cytolysis. The significance of these results is discussed and the hypothesis that synthesis of the maturation producing factor (MPF) requires both energy and protein synthesis is proposed.