Identification of pigment cells during early amphibian development (Triturus alpestris, Ambystoma mexicanum).

The purpose of the present investigation was to provide and apply a methodological manual with which the distribution, patterning and relationship of melanophores and xanthophores can be analyzed during early amphibian development. For demonstration of the methods, which include ultrastructural, histochemical and biochemical approaches, Triturus alpestris and Ambystoma mexicanum (axolotl) embryos are used. These two species differ conspicuously in their larval pigment patterns, showing alternating melanophore bands in horizontal (T. alpestris) and vertical (axolotl) arrangements. With transmission- and scanning electron microscopy melanophores and xanthophores were distinguished by their different pigment organelles and surface structures. The presence of phenol oxidase (tyrosinase) was used to reveal externally invisible or faintly visible melanophores by applying an excess of 3,4 dihydroxy-phenylalanine (dopa). Xanthophores were made visible in fixed and living embryos by demonstrating their pterin fluorescence. In addition, pterins were analyzed by HPLC in embryos before and after pigmentation was visible.

The role of glycosaminoglycans in anuran pigment cell migration.

The migratory pathways of neural crest-derived pigment cells were examined in two anurans, Xenopus laevis and Discoglossus pictus, and correlated with the distribution of glycosaminoglycans (GAG) in the extracellular matrix (ECM) of these pathways. In Xenopus, melanophores in the trunk reach the dermis by initially migrating ventrally, between the neural tube and somites, and then by migrating through the somites to reach the subectodermal space. In Discoglossus, melanophores, iridophores, and xanthophores migrate laterally over the dorsal margin of the somites to reach the dermis. GAG was identified in the light microscope using alcian blue staining and in the electron microscope using ruthenium red staining. The ECM at the dorsal entrance to the lateral pathway in Xenopus and in young Discoglossus (at a stage prior to invasion by pigment cells) is filled with 25-50 nm chondroitin sulphate proteoglycan aggregates. When this ECM in Xenopus is digested in vivo with chondroitinase ABC, melanophores enter the lateral pathway. In older Discoglossus embryos, the migration of pigment cells into the lateral pathway is correlated with increases in the space between the ectoderm and somites and in the number of hyaluronate microfibrils. These observations suggest that chondroitin sulphate proteoglycan in the subectodermal ECM restricts the migration of pigment cells into the lateral pathway by limiting the amount of space for migration and possibly by acting as a less adhesive migratory substratum than the ventral pathway, and that in Discoglossus hyaluronate opens spaces permitting the migration of pigment cells directly over the dorsal margin of the somites.

Comparative behavioral, biochemical and pigmentary effects of MPTP, MPP+ and paraquat in Rana pipiens.

We demonstrate that injections of 1-methyl-4-phenyl-1,2,3,6-tetra-hydropyridine (MPTP), 1-methyl-4-phenyl-pyridinium ion (MPP+) and Paraquat (PQ+) produce in Rana Pipiens different behavioral, biochemical and skin pigmentation changes. MPTP causes in frogs the main symptoms of Parkinsonism (rigidity, akinesia and tremor) and it darkens the skin of animals. It also decreases brain and, less so, adrenal medulla dopamine. These effects are blocked by Pargyline. MPP+ causes the same symptoms but more rapidly. In contrast, skin pigmentation is clearly lightened. Brain and particularly adrenal dopamine reserves are nearly abolished. Pargyline increases these effects. Paraquat, in a cumulative fashion, eventually causes the same behavioral changes and a slight increase in pigmentation. It initially produces an increase in brain and adrenal dopamine concentrations, but later a significant dopamine concentration decrease. Pargyline potentiates these long term effects, blocks the dopamine increase, but reverses the PQ+ effect upon melanin, producing the same depigmentation as MPP+ alone.

Long-term cultivation of amphibian melanophores. In vitro ageing and spontaneous transformation to a continuous cell line.

Pure melanophore populations isolated from the tail skin of the tadpole, Rana catesbeiana, were mass cultured for a period of 2-3 years. All cell lines of amphibian melanophores studied exhibited growth crisis (in vitro ageing) followed by spontaneous transformation to a continuous cell line, as shown by changes in growth characteristics in mass culture and in clone culture, by the appearance of the cells, and by measurements of cell volumes. Even after becoming a continuous cell line, amphibian melanophores continued to have a diploid chromosome number (2n = 26) in three of four cell lines examined. The chromosome mode in one cell line, however, changed to thirty. Measurement of melanin dispersion after the addition of alpha-melanocyte-stimulating hormone suggested that the mechanism for melanin dispersion in melanophores changed during in vitro ageing.

Evidence for rapid structural and functional changes of the melanophore microtubule-organizing center upon pigment movements.

Melanophores of the angelfish, pterophyllum scalare, have previously been shown to display approximately 2,400 microtubules in cells wih pigment dispersed; these microtubules radiate from a presumptive organizing center, the central apparatus (CA), and their number is reduced to approximately 1,000 in the state with aggregated pigment (M. Schliwa and U. Euteneuer, 1978, J. Supramol. Struct. 8:177-190). In an attempt to elucidate the factors controlling this rapid reorganization of the microtubule apparatus, structure and function of the CA have been investigated under different physiological conditions. As a function of the state of pigment distribution, melanophores differ markedly with respect to CA organization. A complex of dense amorphous aggregates and associated fuzzy material, several micrometers in diameter, surrounds the centrioles in cells with pigment dispersed, and numerous microtubules emanate from this complex in a radial fashion. In the aggregated state, on the other hand, few microtubules are observed in the pericentiolar region, and the amount of fibrous material is greatly reduced. These changes in CA morphology as a function of the state of pigment distribution are associated with a marked difference in its capacity to initiatiate the assembly of microtubules from exogenous pure porcine brain tubulin in lysed cell preparations. After complete removal of preexisting microtubules, cells lysed in the dispersed state into a solution of 1-2 mg/ml pure tubulin have numerous microtubules associated with the CA in radial fashion, while cells lysed in the aggregated state nucleate the assembly of only a few microtubules. We conclude that it is the activity of the CA that basically regulates the expression of microtubules. This regulation is achieved through a variation in the capacity to initiate microtubule assembly. Increase or decrease in the amount of dense material, as readily observed in the cell system studied here, seems to be a morphologic expression of such a physiologic function.

Universal acquired melanosis. The carbon baby.

A Mexican child was born white and progressively became black. At the age of 21 months, the whole integument was deep black in the absence of other alterations. Electron microscopy showed a Negroid pattern in the epidermal melanosomes. Among the different forms of progressive mucocutaneous pigmentations previously described in the literature, we were unable to find a similar case.