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.     

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.     

Functional characterization of a receptor for vasoactive-intestinal-peptide-related peptides in cultured dermal melanophores from Xenopus laevis

A receptor for vasoactive-intestinal-peptide (VIP)-related peptides was functionally characterized in a cell line derived from Xenopus melanophores using a recently described microtiter-plate-based bioassay. Activation of the melanophore VIP receptor by VIP or the peptides pituitary-adenylate-cyclase-activating polypeptide (PACAP 38), PACAP 27, and helodermin stimulated intracellular 3'-5' cyclic adenosine monophosphate (cAMP) accumulation and pigment dispersion in the cells. Helodermin, with an EC50 (concentration of peptide inducing half-maximal melanosome dispersion) of 46.5 pM, was the most potent activator of pigment dispersion, followed by PACAP 38 > VIP > PACAP 27. A similar order of potencies was observed for the peptides to induce cAMP accumulation. The responses to VIP agonists were selectively inhibited by the VIP antagonists PACAP-(6-27) and (N-Ac-Tyr(1)-D-Phe2)-growth-hormone-releasing factor[GRF](1-29)-NH2. Taken together, the results suggest that the melanophores express a VIP receptor that shares certain characteristics of, but also differs significantly from, other previously identified VIP receptors.     

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.     

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

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 alpha-melanocyte-stimulating hormone (alpha-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.     

Effect of denervation on hindlimb regeneration in Xenopus laevis larvae

Xenopus laevis larvae at stages 51-57, according to Nieuwkoop and Faber, were subjected to amputation of the right hindlimb or of both limbs at the thigh or the tarsal level, as well as to somatic denervation of the right limb. Larvae at the same stage having undergone amputation of the right limb or of both limbs and sham denervation of the right limb were used as controls. In experimental series I a single denervation of the right limb was performed at the time of amputation. In experimental series II repeated denervations were performed (before, during and after amputation). Results show that in larvae at stages 51-53 subjected to limb amputation at the proximal level (thigh) even repeated denervation of the right limb did not prevent regeneration, although giving rise to various degrees of hypotrophy. In stage-55 larvae partial inhibition of the regenerative process in the right limb was clearly visible only after repeated denervations and amputation at the proximal level. After amputation at the distal level (tarsalia) the regenerative process in the right limb underwent no significant delay with respect to the controls, although the regenerated right limb was hypotrophic. In stage-57 larvae even a single denervation at the time of amputation was enough to inhibit regeneration of the right limb after either proximal or distal amputation. Therefore, in Xenopus laevis larvae, nerve-dependence for hindlimb regeneration takes place proximodistally as the nerve fibers grow in the limb and it gradually undergoes a process of proximodistal differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Effect of background color on the pigmentation of Xenopus laevis larvae

Larvae of Xenopus laevis (stage 39, after hatching) were reared in the 0.95-L (10-cm height; 9.5-cm diameter) containers with all combinations of white, grey, and black color of bottom and walls (side background). The containers were kept in a closed box at 12-hours illumination per day. After two months (stage 55), the number of pigment cells was counted on lateral side of larval body. Both bottom and side background had a significant effect on number of dermal melanophores.     

The Xenopus laevis centrosome aurora/Ipl1-related kinase.

The cDNA encoding the protein kinase pEg2 was originally cloned through a differential screening performed during the early development of Xenopus laevis. pEg2 orthologues were found in various organisms and were classified in a new family of oncogenic mitotic protein kinases named 'aurora/Ipl1-related kinases' after the Drosophila melanogaster gene aurora and the Saccharomyces cerevisiae gene Ipl1. The catalytic activity of pEg2 is necessary for the mitotic microtubule spindle formation in Xenopus laevis egg extracts. The addition of a dominant negative form of pEg2 to in vitro spindle assembly assays leads to monopolar spindles generated by a defect of centrosome separation. In Xenopus cultured cells, pEg2 was confined around the pericentriolar material once centrosomes were duplicated. The centrosome localization does not depend on the presence of microtubules. However, in vitro, the protein binds to taxol-stabilized microtubules independently of its kinase activity. During mitosis the location of the protein changes, in metaphase the kinase localizes on the microtubules at the poles of the mitotic spindle whereas it is not present on astral microtubules. This localization persists until the segregation of the chromosomes is completed. The presence of the kinase on the spindle may reveal another yet unknown function.     

Ultrastructure of microtubules in dermal melanophores and spinal nerve of the Antarctic teleost Pagothenia borchgrevinki

Summary The antarctic teleost, Pagothenia borchgrevinki inhabits the Antarctic Ocean where the water temperature remains around -1.9° C throughout the year. Dermal melanophores of this fish respond within minutes to epinephrine and theophylline with melanosome aggregation and dispersion, respectively. Numerous cytoplasmic microtubules are present in these cells despite the low environmental temperature. In longitudinal profiles, many microtubules are twisted, beaded and sometimes even branched. In cross sections, C-, U-, S-, 6- and other irregularly shaped tubules are observed. Nocodazole partially disrupts microtubules and inhibits epinephrine-induced pigment aggregation. Pigment movements are also prevented by erythro-9-[3-(2-hydroxynonyl)] adenine. Although the participation of these incomplete microtubules in cell motility remains uncertain, the results indicate that this fish has a cold-resistant microtubule system on which melanosome movements depend. Unlike those in melanophores, microtubules in the axons of spinal nerves are of uniform thickness and often contain an electron-dense core in the center.     

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.     

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.     

Pigment migration in the dermal melanophores of amphibian larvae in the interphase and during mitosis

Functioning of the dermal melanophores was studied in the isolated skin of the Rana temporaria and R. esculenta tadpoles at stages 17-21 and 20-24 (after Kopsch). At all stages we studied melanophores exhibited reaction to light. From stage 18 on repeated alternation of pigment dispersion and aggregation was obtained using melanotropins and melatonin. When observing transition of the melanophores from interphase to mitosis, it was found that dividing dermal melanophores could be distinguished due to changes in their appearance shortly before the end of prophase.     

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.