Dermal Melanophores Research Articles

The differentiation of leaf frog melanophores in culture

During early larval development of mexican leaf frog, Pachymedusa dacnicolor, dermal melanophores are typically black, but a few brown melanophores appear about stage 18. During metamorphic climax brown melanophores begin to dominate and by stage 23 they are the exclusive type. The synthesis of the pigment, a red pteridine-dimer, occurred in the melanophores of organ-cultured back skin isolated from tadpoles. The development of this pigmentation in organ culture was independent of thyroxine at least after the onset of metamorphic climax (stage 19). Isolated melanophores in cell culture conditions proliferated only in the presence of MSH, but even these proliferating melanophores, if derived from larvae of stage 21 or younger, showed no indication of pterorhodin synthesis. Melanophores derived from a stage 22 larva and cultured for 18 days synthesized and deposited pterorhodin.

Light-induced pigment aggregation in cultured fish melanophores: spectral sensitivity and inhibitory effects of theophylline and cyclic adenosine-3',5'-monophosphate.

About 20% of xiphophorin fish (Xiphophorus maculatus) dermal melanophores which were cultured in monolayer for 5 to 10 days responded to light directly, exhibiting reversible melanosome aggregation within a few minutes. In order to determine the spectral sensitivity for the light-response, absorbance changes due to the light-induced melanosome aggregation were measured on single melanophores in a range of wavelengths from 370 to 550 nm using a recording microspectrophotometer. The melanophores showed maximum sensitivity to a wavelength of about 410 nm and very little to those longer than 500 nm. 10(-4) to 10(-2) M theophylline and 10(-4) M cAMP had inhibitory effects on the light-response.

Vertical banding evoked by electrical stimulation of the brain in anaesthetized green sunfish, Lepomis cyanellus, and bluegills, Lepomis macrochirus.

A pattern of dark vertical bands is a characteristic agonistic display in the green sunfish, Lepomis cyanellus and the bluegill, L. macrochirus. The rapidity with which the display can appear and disappear indicates that it is neurally controlled. Electrical stimulation of the brain was carried out in anaesthetized green sunfish and bluegills to map those regions from which this colour change can be elicited. Banding was evoked by stimulation of sites near the midline in the preoptic area, ventral thalamic-dorsal hypothalmic transition zone, the midbrain tegmentum (just dorsal to the nucleus prerotundus pars medialis), in and near the torus semicricularis, in the basal midbrain (region of the crossing tectobulbar tracts), and in the rostral basomedial medulla. A 'transition' zone was located basally in the middle medulla, caudal to which only paling was evoked. Areas found to be negative for evoked banding included the telencephalic lobe, the inferior lobe of the hypothalamus, the optic tract, the optic tectum, the body and valvula of the cerebellum and the caudal medulla. It is postulated that the vertical banding pattern is made up of a separate, selectively controlled system of dermal melanophores. The possible neural mechanisms controlling banding are discussed.

Differential chromatic activity of melanophores in integumentary patterns of winter flounder (Pseudopleuronectes americanus Walbaum)

Winter flounder chromatophores include three major melanophore systems with differences in effector distribution and chromatic behavior. Response characteristics suggest that each of these pattern components represents a different neuroeffector system and that there may he prolonged hormonal control. One pattern component has the capacity for rapid melanosome dispersion and a second has the capacity for rapid melanosome aggregation on different backgrounds. Chromatic behavior of the third component is not necessarily background related. Although epidermal and dermal melanophore responses both exhibit rapid and slow phases, greater degrees of melanosome aggregation occur in epidermal melanophores and a higher stimulation threshold for their melanosome dispersion is indicated.

Ultrastructural study of the cutaneous pigment cells of wild-type and albinistic bullfrogs, Rana catesbeiana

Ultrastructural comparisons were made of cutaneous pigment cells from analogous skin areas of variant albinistic and control wild-type bullfrogs, Rana catesbeiana . In the epidermis, the wild-type melanocytes contained numerous mature melanosomes whereas the albinistic epidermal melanocytes demonstrated retarded melanogenesis with premelanosome stages dominant. In the dermal chromatophore unit of both frogs, no morphological differences were observed in the xanthophores, but significant morphological distinctions were noted in the melanophores and iridophores. The wild-type dermal melanophores contained numerous mature melanosomes whereas the albinistic dermal melanophore demonstrated retarded melanogenesis similar to that found in its epidermal melanocyte. The albinistic iridophores not only had reduced reflecting platelet areas, but also the platelets were aberrant in morphology compared to the rectangular shape of the reflecting platelets in the wild type. The mutant reflecting platelets were small, spherical, and highly irregular in shape. It is suggested that the development of each type of albinistic melanocyte, melanophore, and iridophore might occur if defective MSH-membrane-receptors altered or diminished the cell response to MSH during pigment cell ontogeny.

Melanophore distribution within the integumentary tissues of two teleost species, Pseudopleuronectes americanus and Gasterosteus aculeatus form leiurus

In winter flounder, dermal melanophores are prominent chromatic components in the skin of the entire upper surface, and their arrangement is affected by imbrication of the ctenoid scales. Epidermal melanophores are also prominent over most of the body upper skin, scale imbrication having relatively little effect on their arrangement. Epidermal melanophores occur only sparsely on the fins and head skin, and are virtually absent from white spots of flounder disruptive patterns. In stickleback, epidermal melanophores were not observed. Melanophores with thin processes form a superficial dermal layer in the dorsal skin of the head and body of stickleback, and along the fin dermal supports. Melanophores with broad, wedge-like, processes form an extensive and continuous deep dermal layer in most of the body skin, which lacks scales, and on the operculum. Imbrication of the small number of lateral bony plates behind the operculum in leiurus sticklebacks does not greatly affect the melanophore arrangement. The skin structure of both species shows sexual dimorphism, but the respective melanophore layers are strongly developed in each sex.

The estimation of melanophore responses in a pleuronectid fish

A description is given of a simple method of estimating physiological responses of epidermal and dermal melanophores in a pleuronectid fish.

The Effect of The Divalent Cation Ionophore A23187 on Amphibian Melanophores and Iridophores

Calcium ions are required in the medium for the full darkening action of melanocyte-stimulating hormone, but not for its second messenger, cyclic adenosine monophosphate, on isolated frog skin. Thus, the possible effect of the divalent cation ionophore A23187 was studied, using the skin of Rana pipiens. It is a potent darkening agent over the range of 1-2 micrometer, as measured by reflectance change and microscopic observation. During the darkening response of A23187, melanosome dispersion takes place in both dermal and epidermal melanophores, as well as aggregation of iridophore reflecting platelets. The darkening is slowly reversible in Ringer's solution, thus it is not due to toxicity. The darkening is partially dependent on the presence of calcium in the medium under certain conditions, suggesting that the response involves both the uptake and release of calcium ions. Cytochalasin B reversibly inhibits the darkening produced by A23187. Since this drug is known to bring about the breakdown of microfilaments, its inhibitory action is in accord with the concept that the ionophore is stimulating microfilament function. A23187 administered to tissue cultured embryonic salamander (Ambystoma maculatum) melanophores produced an irreversible rounding up of the cells, suggesting a toxic effect. The results with frog skin are interpreted as supporting the concept that the action of melanocyte-stimulating hormone involves the production of pigment granule movements as a result of the interaction of calcium ions with intracellular microfilaments and possible also the breakdown of microtubules. The mobilization of calcium ions could be brought about by cyclic adenosine monophosphate.

Ultrastructural analysis of gene interaction and melanosome differentiation in the retinal pigment cells of the albino goldfish.

The genotype of albino goldfish is represented by pp,cc, and the non-albino fish is PP,CC. The P gene begins to control the melanosome formation of retinal pigment cells at St-21; and the C, which is epistatic to P and p, governs melanosome formation in the dermal melanophores and in retinal pigment cells after St-22. Then in the pp,CC fish, between St-21 and St-22, melanosomes of the pigmented retina are albino in type, but after St-22 they become non-albino in type. From ultrastructural observations on the melanosomes of these late-melanizing fish, it is apparent that the imcompletely melanized albino pigment granules can differentiate into normal melanosomes under the control of the C gene. The processes of melanosome maturation in the late-melanizing goldfish are evident from these observations.

Heterochromatin dispersion and blebbing of the nuclear membrane induced by melanophore stimulating hormone.

Stimulation (in vivo and in vitro) of dermal melanophores of the leaf frog, Agalychnis dacnicolor, by melanophore stimulating hormone (MSH) elicits two responses in addition to the dispersion of melanosomes: (1) dispersion of heterochromatin; and (2) blebbing of the outer membrane of the nuclear envelope. The latter leads to the formation of dilated rough endoplasmic reticulum with the involvement of 80–100 A microfilaments. N6,O2-dibutyryl adenosine 3′,5′-monophosphate (db-cAMP) elicits a similar response. Actinomycin D prevents both heterochromatin dispersion and membrane blebbing while cytochalasin B (CB) prevents only the latter.

On the development of chicken adenohypophyseal melanophore-expanding activity.

Melanophore-expanding activity (MEA) was detectable at least by stage 30 of Hamburger and Hamilton (6–6.5 incubation days) in 53 whole adenohypophyseal anlagen (plus mesenchyme) from domesticated Gallus gallus embryos. This was estimated from responses to adenohypophyseal implants next to dermal melanophores of intact Rana temporaria tadpoles, as compared with control implants of embryonic myelencephalic tissue. Analyses of 27 implants of adenohypophyseal cephalic- or caudal-region tissue alone indicated that MEA was associated mostly with cephalic regions. This association started at least by stage 35 (8–9 incubation days) and continued until the 1st day post hatching, as compared with control implants of embryonic eyelid tissue.

Fine structure of nerve-melanophore contacts in the angelfish Pterophyllum scalare.

Contacts between small unmyelinated nerve fibres and dermal melanophores of the angelfish, Pterophyllum scalare, exhibit several features characteristic of synapses, including small synpatic vesicles and dense core vesicles, a narrow synaptic cleft, electron-dense material at the postsynaptic membrane (cell membrane of the melanophore) and, occasionally, presynaptic densities. An analysis of serial thin sections shows that the synapses described here represent varicosities of an otherwise more or less straight nerve fibre. A single axon thereby may form several en passant synapses with a single melanophore. It is suggested that the synaptic contacts described here not only represent sites of transmitter release but also play a role as sites of firm attachment between nerves and melanophores which guarantee a stable arrangement of nerve fibres and melanophores.

Melatonin excretion of man and rats: effect of time of day, sleep, pinealectomy and food consumption.

Pineal function was assessed in human subjects by measuring the excretion of melatonin. The hormone was extracted from the urine by adsorption on a nonionic polymeric resin and then elution with organic solvents; its concentration was determined with a bioassay based ontthe dermal melanophore response of larval anurans to melatonin in their bathing medium. Melatonin excretion among healthy, adult volunteers was 5- to 7-fold greater during the hours of sleep, darkness, and recumbancy (23:00–07:00 h) than during the active, waking hours (07:00–15:00 h or 15:00–23:00 h). When 2 subjects slept only during the daylight at odd, 8-hour intervals, their melatonin excretion was also greatest during their time of sleep. Three bedfast fracture patients (for whom sleep data were not available) failed to display melatonin rhythms. A new radioimmunoassay for melatonin was investigated in preliminary studies with experimental animals and was found to be expedient and sensitive; it was not as specific as the bioassay, however. Both analytical methods were used in studies on intact and pinealectomized rats, and the findings suggest that rhythmic melatonin excretion persists (although at a reduced amplitude) in the absence of the pineal. Fasting in the pinealectomized animal abolished the day/night variation in urinary melatonin, but did not have this effect in intact rats.

Ultrastructure of the integumental melanophores of the coelacanth, Latimeria chalumnae.

The integumental melanophores of Latimeria chalumnae were studied by light and electron microscopy. The epidermal melanophore located in the mid-epidermis consists of a round perikaryon with long slender dendrites extending into epidermal cells and intercellular spaces. The dermal melanophores occur in the loose dermal matrix underlying a relatively thick layer of collagen fibers. The dermal melanophores are usually flattened and their dendrites lie parallel to the collagen layer. Both epidermal and dermal melanophores contain oval, electron-opaque melanosomes, large mitochondria, agranular vacuoles of endoplasmic reticulum and microtubules. Microfilaments and RNP particles are less conspicuous. While the peripheral cytoplasm of both dermal and epidermal melanophores is filled with a large number of melanosomes, the perinuclear cytoplasm of many dermal melanophores is occupied by premelanosomes in various stages of differentiation, and that of the epidermal melanophore contains numerous large vacuoles. Despite the scarcity of epidermal melanophores, the epidermal melanin unit is present in the form of melanosome complexes. In addition, the melanophores of Latimeria possess the basic characteristics common to other vertebrates, but they more closely resemble those of lungfish and other aquatic vertebrates.

Ultrastructure of the integumental melanophores of the South Americal lungfish (Lepidosiren paradoxa) and the African lungfish (Protopterus sp.).

The integumental melanophores of two genera of lungfish, Lepidosiren paradoxa and Protopterus sp. were examined by light and electron microscopy. Both species possess both epidermal and dermal melanophores with fine structural characteristics basically similar to those of other vertebrates. The epidermal melanophores of both species are located in the intermediate epidermis, and possess thin perikarya containing round nuclei, and slender dendrites extending into the nearby intercellular spaces. The dermal melanophores occur immediately beneath the basement membrane, and possess flat perikarya and dendrites running horizontally between the collagen fibers of the dermis. The integument of both species does not possess an epidermal melanin unit or a dermal chromatophore unit. As in other vertebrates, each melanophore contains numerous oval, electron-opaque melanosomes, relatively large mitochondria, vacuolar endoplasmic reticula, and groups of RNP particles. Although micro filaments running randomly between other organelles occur regularly, microtubules were not demonstrated. Premelanosomes at various stages of differentiation were best illustrated in the dermal melanophores of Protopterus, and it is concluded from the observation of their fine structure that the morphological development of lungfish melanosomes closely parallels that of higher vertebrates. On the basis of melanophore morphology, Lepidosiren and Protopterus appear to be more closely related to each other than to Neoceratodus.

Ultrastructure of the integumental melanophores of the Australian lungfish, Neoceratodus forsteri.

The integumental melanophores of Australina lungfish, Neoceratodus forsteri, were examined by light and electron microscopy and found to possess essentially the same structural characteristics observed in other vertebrates. The epidermal melanophores are located in the intermediate epidermis and possess round perikarya and slender dendrites extending into nearby intercellular spaces. The dermal melanophores are found immediately below the basement membrane as well as in the deeper dermis. These cells possess flattened nuclei and dendrites running parallel to the basement membrane. Each melanophore contains numerous oval or elliptical, intensely electron-dense melanosomes, relatively large mitochondria, systems of vacuolar endoplasmic reticulum, groups of free RNP particles, and some microfilaments. Only a few, short microtubules could be demonstrated in the perinuclear cytoplasm of the dermal melanophore, while a relatively large number of late premelanosomes are found both in perikarya and dendritic processes of epidermal melanophores. These premelanosomes exhibit a particulate internal structure in cross section. Both melanosomes and premelanosomes occur singly in the cytoplasm of epidermal cells, thereby confirming the existence of the epidermal melanin unit in the lowest vertebrates thus far examined electron microscopically.

In vitro response of goldfish (Carassius auratus L.) dermal melanophores to cyclic 3',5'-nucleotides, nucleoside 5'-phosphates and methylxanthines.

AbstractcAMP, dbcAMP, cCMP, cGMP, theophylline and caffeine caused reversible melanosome dispersion within 5 minutes at 10 mM in the dermal melanophores of the black goldfish, Carassius auratus L. cTMP, cUMP, 5′‐AMP, 5′‐CMP, 5′‐GMP, 5′‐TMP, and 5′‐UMP did not produce melanosome dispersion or aggregation in this melanophore system. cAMP was the most effective nucleotide in the induction of melanosome dispersion; at 10 mM, cGMP and at 5 mM, dbcAMP were the least effective of those nucleotides inducing melanosome dispersion. At the 10 mM level dbcAMP required 30 minutes to evoke the same degree of melanosome dispersion as 5 minutes cAMP treatment. Theophylline was more effective than caffeine in eliciting melanosome dispersion. At 1 mM, theophylline and caffeine first induced melanosome dispersion which was followed by aggregation in the course of the 30 minute test period. These reactions suggest both a high melanophore phosphodiesterase activity and competitive inhibition of phosphodiesterase by theophylline and caffeine. Induction of melanosome dispersion by several cyclic 3′,5′‐nucleotides suggest multi‐nucleotide control of melanosome dispersion. These findings also support a proposed mechanism of prostaglandin induced melanosome dispersion as well as the “second messenger” hypothesis.

Response of dermal melanophores to epinephrine after removal of the epidermal barrier

1. 1. Comparison of methods for selectively removing epidermis from goldfish integument demonstrated that the use of EDTA provided efficient epidermis removal, excellent melanophore viability and good dermal cell preservation. 2. 2. Photometric measurement of melanophore response to epinephrine and to goldfish melanophore Ringer solution permitted quantitation of the degree of melanosome dispersion. 3. 3. Removal of the epidermis produced a tenfold increase in melanophore sensitivity to epinephrine and decreased post-treatment recovery rate. Thus, the epidermis is a diffusion barrier of considerable magnitude. 4. 4. Lipophores (xanthophores) and iridophores were unresponsive to epinephrine treatment (10 μg/ml) indicating that epinephrine is not involved in the normal hormonal control of these pigment cells.

Morphogenesis of the dorsal pigmentary pattern in wild-type and mutant Rana pipiens

The transition from larval to adult pigmentary patterns during metamorphosis of wild-type. burnsi, and kandiyohi R. pipiens is described. Larval fusiform epidermal melanocytes form a pattern that exactly corresponds to the adult spotting pattern. It is concluded that the larval epidermal pattern expresses a “prepattern” in the larval tissue for the adult pattern. This “prepattern” is visible in kandiyohi, but not in burnsi, tadpoles. The role of the extracellular environment in pigmentary pattern determination is discussed. Gradual changes in all chromatophore densities accompany larval development, while abrupt changes accompany metamorphic climax. There is a net increase in all chromatophore densities by the completion of metamorphosis. Kandiyohi density changes differ quantitatively but not qualitatively from those of wild type. In both wild type and kandiyohi, differentiation of many new dermal melanophores in presumptive spot regions effects expression of the adult spotting pattern. The relative roles of chromatophore differentiation and mitosis in pattern expression, and the hormonal control thereof, are discussed.

Formula omitted] effects of prostaglandins upon melanosome dispersion in the skin of black goldfish, [formula omitted] L.

Prostaglandins affect physiologic changes in teleosts as demonstrated by in vitro evocation of melanosome dispersion in dermal melanophores of the skin of black goldfish, Carassius auratus L. The prostaglandins (10 to 100 μg/ml) induce dose dependent increases in melanosome dispersion which are reversed by epinephrine (10 μg/ml). Prostaglandins A 2 and B 2 are the most effective in eliciting melanosome dispersion while prostaglandin F 1α and F 2α are least effective. The presence of a C-9 carbonyl group appears to be important in prostaglandin induced melanosome dispersion. The prostaglandins may evoke their response by dissociating a calcium-ATP complex in the membrane allowing ATP to serve as a substrate for adenyl cyclase.