Pigmentary system of the adult alpine salamander Salamandra atra aurorae (Trevisan, 1982).

The pigmentary system of skin from adult specimens of the amphibian urodele Salamandra atra aurorae was investigated by light microscope, electron microscope, and biochemical studies. Yellow (dorsum and head) and black (flank and belly) skin was tested. Three chromatophore types are present in yellow skin: xanthophores, iridophores, and melanophores. Xanthophores are located in the epidermis whereas iridophores and melanophores are found in the dermis. Xanthophores contain types I, II, and III pterinosomes. Some pterinosomes are very electron-dense. Black skin has a single type of chromatophore: the melanophores. Some melanophores are located in the epidermis. In contrast to the dermal melanophores, these present, in addition to typical melanosomes, organelles with different morphology and vesicles having a limiting membrane and containing little amorphous material. Both skin types present some pteridines and flavins, though they are qualitatively and quantitatively more abundant in yellow skin extracts.

Quantitative in vitro assay for crustacean chromatophorotropins and other pigment cell agonists.

An in vitro crustacean (freshwater shrimp, Macrobrachium potiuna) erythrophore bioassay for chromatophorotropins and other pigment cell agonists is described. The present assay is a quantitative method that determines the pigment responses with the aid of an ocular micrometer. The pigment granules within the erythrophores are dispersed out into the dendritic processes of the cells when the isolated carapace is placed in physiological solution. This bioassay provides, therefore, a method for measuring the response of the pigment cells to aggregating agents such as pigment concentrating hormone (PCH). This bioassay is sensitive to PCH at a concentration as low as 3 x 10(-12) M. Calcium ionophore A23187 mimics the actions of PCH, but, unlike the hormone, the ionophore-induced pigment aggregation is irreversible after physiological solution rinses. Therefore, chromatophorotropic activities of pigment dispersing agents, such as pigment dispersing hormones (PDH), can be determined on ionophore-treated erythrophores. The potencies of alpha-PDH and beta-PDH show a threefold difference (not significant). Because of its convenience and its ability to make an objective determination of the bidirectional pigment movements within erythrophores, this bioassay is a suitable method for further structure-activity studies of the various chromatophorotropins and their analogs.

Immunofluorescence evidence for cytoskeletal rearrangement accompanying pigment redistribution in goldfish xanthophores.

Immunofluorescence and phase-contrast microscopic studies of goldfish xanthophores with aggregated or dispersed pigment show two unusual features. First, immunofluorescence studies with anti-actin show punctate structures instead of filaments. These punctate structures are unique for the xanthophores and are absent from both goldfish dermal non-pigment cells and a dedifferentiated cell line (GEM-81) derived from a goldfish xanthophore tumor. Comparison of immunofluorescence and phase-contrast microscopic images with electron microscopic images of thin sections and of Triton-insoluble cytoskeletons show that these punctate structures represent pterinosomes with radiating F-actin. The high local concentration of actin around the pterinosomes results in strong localized fluorescence such that, when the images have proper brightness for these structures, individual actin filaments elsewhere in the cell are too weak in their fluorescence to be visible in the micrographs. Second, whereas immunofluorescence images with anti-tubulin show typical patterns in xanthophores with either aggregated or dispersed pigment, namely, filaments radiating out from the microtubule organizing center, immunofluorescence images with anti-actin or with anti-intermediate filament proteins show different patterns in xanthophores with aggregated versus dispersed pigment. In cells with dispersed pigment, the punctate structures seen with anti-actin are relatively evenly distributed in the cytoplasm, and intermediate filaments appear usually as a dense perinuclear band and long filaments elsewhere in the cytoplasm. In cells with aggregated pigment, both intermediate filaments and pterinosomes with associated actin are largely excluded from the space occupied by the pigment aggregate, and the band of intermediate filaments surrounds not only the nucleus but also the pigment aggregate. The patterns of distribution of the different cytoskeleton components, together with previous results from this laboratory, indicate that formation of the pigment aggregate depends at least in part on the interaction between pigment organelles and microtubules. The possibility that intermediate filaments may play a role in the formation/stabilization of the pigment aggregate is discussed.

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.

Evidence for intermediate filaments in squirrelfish erythrophores of Holocentrus ascensionus (Rufus).

We have documented the presence of intermediate filaments (IF) in cultured erythrophores of the squirrelfish Holocentrus ascensionus (Rufus). SDS-PAGE and Western blots with monoclonal antibodies T11 and R12 demonstrated that isolated IF consisted of a pair of polypeptides of 54 and 52 kDa. Immunofluorescent studies revealed that the two proteins formed prominent radially oriented IF networks in erythrophores. Immunoelectron microscopic studies showed that the IF were distributed in a "spider-web"-like network of filaments which occasionally intersected with the microtubule surfaces. The IF proteins also were found in fish iridiphores but not in fish epithelial cells which cocultured with the chromatophores.

Chromatophore motoneurons in the brain of the squid, Lolliguncula brevis: an HRP study.

The location of the motoneuron somata controlling activity of the chromatophore muscles was studied in the squid Lolliguncula brevis. Retrograde transport of horseradish peroxidase from injection sites in the skin or in the mantle muscle established that the chromatophore motoneurons are situated in the subesophageal mass of the brain while at least some of the mantle muscle motoneurons are in the stellate ganglia. Motoneurons to chromatophores in the mantle have their somata in the posterior subesophageal mass, mainly in the chromatophore or fin lobes. Motoneurons to chromatophores in the head are located in the anterior pedal lobes and those to the chromatophores in the arms project mainly from the anterior chromatophore lobes. However, some neurons in the posterior chromatophore lobes project to the head or arm regions. A few cells in both the anterior and posterior chromatophore lobes project contralaterally. Somata in other lobes of the subesophageal mass are also labelled by injections in the skin or in the mantle muscle. Evidence presented here suggests that some of the neurons labelled outside the chromatophore lobes are chromatophore motoneurons.

The pigmentary system of developing axolotls. I. A biochemical and structural analysis of chromatophores in wild-type axolotls.

A biochemical and transmission electron microscopic description of the wild-type pigment phenotype in developing Mexican axolotls (Ambystoma mexicanum) is presented. There are three pigment cell types found in adult axolotl skin - melanophores, xanthophores and iridophores. Both pigments and pigment cells undergo specific developmental changes in axolotls. Melanophores are the predominant pigment cell type throughout development; xanthophores occur secondarily and in fewer numbers than melanophores; iridophores do not appear until well into the larval stage and remain thereafter as the least frequently encountered pigment cell type. Ultrastructural differences in xanthophore organelle (pterinosome) structure at different developmental stages correlate with changes in the pattern of pteridine biosynthesis. Sepiapterin, a yellow pteridine, is present in larval axolotl skin but not in adults. Riboflavin (also yellow) is present in minimal quantities in larval skin and large quantities in adult axolotl skin. Pterinosomes undergo a morphological "reversion" at some point prior to or shortly after axolotls attain sexual maturity. Correlated with the neotenic state of the axolotl, certain larval pigmentary features are retained throughout development. Notably, the pigment cells remain scattered in the dermis such that no two pigment cell bodies overlap, although cell processes may overlap. This study forms the basis for comparison of the wild type pigment phenotype to the three mutant phenotypes-melanoid, axanthic and albino-found in the axolotl.

The pigmentary system of developing axolotls. II. An analysis of the melanoid phenotype.

The melanoid mutant in the Mexican axolotl (Ambystoma mexicanum) is analysed with respect to the differentiation of pigment cells. Pigment cells were observed with the transmission electron microscope in order to determine any unusual structural characteristics and to determine what happens to each of the cell types as development proceeds. Chemical analysis of pteridine pigments was also carried out, and changes in pteridine biosynthesis were found to correlate well with changes in xanthophore morphology and number. In melanoid axolotls, as development proceeds, melanophore numbers increase, xanthophores decrease, and iridophores fail to differentiate at all. This is considered to result from: (a) conversion of xanthophores (that are present in young larvae) to melanophores; (b) the gradual programming of the majority of chromatoblasts to become, exclusively, melanophores, and (c) the failure of some chromatoblasts (possibly iridoblasts) to differentiate altogether. The ultrastructural and chemical evidence presented in this study is compared to similar data for wild-type axolotls, and a mechanism regarding how the melanoid gene might act is suggested.

Resonance Raman study of the primary photochemistry of visual pigments. Hypsorhodopsin.

We report here the first resonance Raman results of octopus hypsorhodopsin, a species formed photochemically at very low temperatures from visual pigments. A pump-probe technique was used to obtain Raman spectra from samples at 12 degrees K whose photostationary state mixtures were either hypsorhodopsin rich or hysorhodopsin poor. The data strongly suggest that the Schiff-base linkage between the chromophore of hysorhodopsin and apoprotein is protonated. Further, the results suggest that hypsorhodopsin's chromophore is in some torsionally distorted conformation, possibly having torsional departures from an all-trans isomeric form.

Intermediate filaments in the cytoskeletons of fish chromatophores.

When fish pigment cells (melanophores, erythrophores) are lysed by a modified Kleinschmidt method on a buffer-air interface and examined by electron microscopy, large numbers of intermediate filaments are observed. The intermediate filament networks are distinct from actin and tubulin, and entrap the pigment as determined by stereo viewing of freeze-dried rotary-shadowed specimens. During lysis, under conditions that do not preserve actin filaments or microtubules, the area covered by dispersed pigment granules reaches a maximum size and remains stable for many minutes, suggesting that intermediate filaments are responsible for holding the pigment in position and preventing further cytoplasmic dispersion. These observations demonstrate that fish pigment cells contain large numbers of intermediate filaments and suggest that they may be important for coordinating pigment granule movement.

The role of microtubules in cell shape and pigment distribution in spreading erythrophores.

In order to investigate the role of microtubules in determining overall shape of the cell and distribution of pigment granules, a correlative whole cell electron microscopic and immunofluorescence light microscopic study was done of microtubule distribution during spreading of cultured erythrophores from scales of the squirrel fish, Holocentrus. After dissociation from the scale and immediately after attachment, erythrophores are round with long thin processes containing bundles of microtubules with associated pigment granules. In time these processes attach, and elongate, and cytoplasmic ground substance fills areas between microtubule bundles, preceding the appearance of microtubules in these areas of the cell. By 8 h cells are fully spread (star-shaped), prominent microtubule bundles have disappeared, and microtubules (along with pigment granules) are more evenly dispersed throughout the cell. After 24 h cells have increased in size, in number of microtubules, and in the length of preexisting tubules. The sequence of events in normal spreading is altered if cytochalasin D, cycloheximide, or nocodazole is included in the culture medium. Cytochalasin D, a microfilament-disrupting drug, prevents attachment and spreading, but allows elongation to occur. Cycloheximide, a protein synthesis inhibitor, allows for attachment and elongation, but no spreading. Nocodazole, a microtubule-disrupting drug, allows for attachment and spreading, but an irregular cell outline is the result. Even though spreading can occur without them, it is concluded that a normal number and distribution of microtubules are required for the development of normal cell shape and pigment distribution.

Carotenoid and pterin pigment localization in fish chromatophores.

The classical sulfuric acid method for the histochemical detection of carotenoids has been adapted to give a reliable cytological localization of these compounds in fish chromatophores. This procedure consists mainly in fixing skin fragments in glutaraldehyde and dehydrating in a 50% solution of glycerin followed by exposure to air. It is essential that the preparation permit direct contact of the sulfuric acid with the pigment cells. Under these conditions, carotenoid containing cells stain green or blue. When associated with the extraction of the carotenoids by acetone, the procedure permits the distinction between pterin and carotenoid in fish chromatophores.

Biochemical characterization of crystals from the dermal iridophores of a chameleon Anolis carolinensis.

The biochemical characteristics of dermal iridophore crystals from Anolis carolinensis have been investigated. Iridophores isolated by collangenase-hyaluronidase treatment were sonicated and their contents fractionated through sucrose. Pure iridophore crystals so obtained were examined by chromatography and electron diffraction. They were found to be pure hydrated crystalline form. The suggestion is made that the subcrystalline structure of this guanine does not play a role in color production by the iridophore.