Ultrastructural changes in the dermal chromatophore unit of Hyla arborea during color change

Summary The structural changes in the chromatophores of Hyla arborea related to changes in skin color were studied by electron microscopy and reflectance microspectrophotometry. During a change from a light to a darker green color, the melanosomes of the melanophores disperse and finally surround the iridophores and partly the xanthophores. The iridophores change from cup-shape to a cylindrical or conical shape with a simultaneous change in the orientation of the platelets from being parallel to the upper surface of the iridophores to being more irregular. The xanthophores change from lens-shape to plate-shape. The color change from green to grey seems always to go through a transitional black-green or dark olive green to dark grey. During this change the xanthophores migrate down between the iridophores, and in grey skins they are sometimes found beneath them. The pterinosomes gather in the periphery of the cell, while the carotenoid vesicles aggregate around the nucleus. The iridophores in grey skin are almost ball-shaped with concentric layers of platelets. A lighter grey color arises from a darker grey by an aggregation of melanosomes. The chromatophore values previously defined for Hyla cinerea are applicable in Hyla arborea, and the ultrastructural studies support the assumptions previously made to explain these values.The author wishes to thank Drs. P. Budtz, J. Dyck and L.O. Larsen for valuable discussions and J. Dyck for kindly providing the spectrophotometer granted him by the Danish National Science Foundation. The skilled technical assistance of Mrs. E. Schiøtt Hansen is gratefully acknowledged. Permission was granted by the Springer-Verlag to republish the illustrations of W.J. Schmidt (1920)     

Hormone-induced pigment translocations in amphibian dermal iridophores, in vitro: changes in cell shape.

Hormone-induced pigment translocation studies were conducted at both the light and electron microscopic levels on cultured dermal iridophores from the Mexican leaf frog, Pachymedusa dacnicolor. Two distinct types of dermal iridophores were characterized which differed in (1) their in vivo locations, (2) their overall morphologies in vitro, (3) their responses to alpha-MSH, ACTH, c-AMP or theophylline, (4) their physical alterations of light, and (5) certain ultrastructural features. One iridophore (Type I) was found to be physiologically responsive to the above hormones or agents by a reversible retraction of cellular processes and a thickening of the cell body, an event which is inhibited by cytochalasin B. The other iridophore (Type II) appeared to be unresponsive. Type I iridophores contain cube-like pigmentary organelles, refractosomes, while Type II iridophores contain larger, bar-shaped refractosomes. In addition, both iridophore types contain 60 and 100 A microfilaments as well as microtubules. By in large, micorfilaments were found within microvilli, beneath and parallel to the plasma membrane and in the perinuclear region. Occasionally, bundles of 100 A microfilaments were found between layers of refractosomes in Type I iridophores. These results are discussed in relation to hormone-induced changes in cell shape.     

Physiological color change in squid iridophores

Evidence is presented that changes in the optical properties of active iridophores in the dermis of the squid Lolliguncula brevis are the result of changes in the ultrastructure of these cells. At least two mechanisms may be involved when active cells change from non-iridescent to iridescent or change iridescent color. One is the reversible change of labile, detergent-resistant proteinaceous material within the iridophore platelets, from a contracted gel state (non-iridescent) to an expanded fluid or sol state when the cells become iridescent. The other is a change in the thickness of the platelets, with platelets becoming significantly thinner as the optical properties of the iridophores change from non-iridescent to iridescent red, and progressively thinner still as the observed iridescent colors become those of shorter wavelengths. Optical change from Rayleigh scattering (non-iridescent) to structural reflection (iridescent) may be due to the viscosity change in the platelet material, with the variations in observed iridescent colors due to changes in the dimensions of the iridophore platelets.     

The Blue Coloration of the Common Surgeonfish, Paracanthurus hepatus-II. Color Revelation and Color Changes

Measurements of spectral reflectance from the sky-blue portion of skin from the common surgeonfish, Paracanthurus hepatus, showed a relatively steep peak at around 490 nm. We consider that a multilayer thin-film interference phenomenon of the non-ideal type, which occurs in stacks of very thin light-reflecting platelets in iridophores of that region, is primarily responsible for the revelation of that hue. The structural organization of the iridophore closely resembles that of bluish damselfish species, although one difference is the presence of iridophores in a monolayer in the damselfish compared to the double layer of iridophores in the uppermost part of the dermis of surgeonfish. If compared with the vivid cobalt blue tone of the damselfish, the purity of the blue hue of the surgeonfish is rather low. This may be ascribable mainly to the double layer of iridophores in the latter since incident lightrays are complicatedly reflected and scattered in the strata. The dark-blue hue of the characteristic scissors-shaped pattern on the trunk of surgeonfish is mainly due to the dense population of melanophores, because iridophores are only present there in a scattered fashion. Photographic and spectral reflectance studies in vivo, as well as photomicrographic, photo-electric, and spectrometric examinations of the state of chromatophores in skin specimens in vitro, indicate that both melanophores and iridophores are motile. Physiological analyses disclosed that melanophores are under the control of the sympathetic nervous and the endocrine systems, while iridophores are regulated mainly by nerves. The body color of surgeonfish shows circadian changes, and becomes paler at night; this effect may be mediated by the pineal hormone, melatonin, which aggregates pigment in melanophores.     

Rapid integumental color changes due to novel iridophores in the chameleon sand tilefish Hoplolatilus chlupatyi

The wavelength of the light reflected from iridophores depends on the thickness and the spacing of intracellular reflecting platelets. Here, we show that the rapid color change from blue to red of the chameleon sand tilefish Hoplolatilus chlupatyi is mediated by adrenergic stimulation of a novel type of iridophore in which reflecting platelets are concentrated selectively in the periphery of the cell, near the plasma membrane. The color changes are not only observed in vivo but also in pigment cells of isolated scales which respond to increases in K⁺ ion concentrations in 0.5 s and to addition of norepinephrine within 1 s. The norepinephrine effect can be blocked by addition of the alpha‐adrenergic antagonist phentolamine. The results suggest that adrenergic stimulation leads to changes in reflecting platelet organization in Hoplolatilus chlupatyi iridophores and represents the major mediator of the rapid color change in this fish in vivo.     

Transformation of amphibian iridophores into melanophores in clonal culture.

Iridophores isolated from bullfrog tadpoles were successfully cloned. In primary culture, the iridophores showed contraction of cell bodies by the addition of alkali-treated ACTH. The disappearance of reflecting platelets occurred in proliferating iridophores and many small black melanin granules were synthesized in the cells. The chromatophores now showed melanin dispersion by the addition of the above hormone. The findings suggest that iridophores transform into melanophores in vitro.     

Microtubular apparatus of melanophores: three dimensional organization

Microtubular organization in the melanophores of the angelfish, Pterophyllum scalare, has been studied by serial thin sectioning. The course of microtubules has been followed in sets of transverse serial sections taken from the centrosphere and a segment of a cell process, respectively. Microtubules arise from a prominent zone in the cell center, the central apparatus, which is composed of numerous, small, electron-dense aggregates. the number of these loosely distributed densities is highest in the center of the centrosphere, but they may also be found at its periphery. Microtubules insert into, or becomes part of, the dense material, or at least start in its vicinity. Dense aggregates may be separated from centrioles by several micrometers rather than only being closely associated with these organelles. At some distance from the organizing zone, most of the microtubules gradually assume a cortical arrangement, i.e., take a course within about 100 nm of the limiting membrane. Serial sections were used to trace all microtubules within a 6μm-long segment of a cell process. 94 percent of the microtubules observed in this segment run its entire length; it is conceivable, therefore that a considerable number of microtubules extend between the initiating site in the centrosphere and the outermost cell region. A three-dimensional model of the 6μm-long segment reveals that, despite changes in the cell process outline, microtubules maintain a strictly cortical arrangement which gives the impression of a microtubule "palisade" lining the cortex of the cell process. The features of the microtubular apparatus of angelfish melanophores are discussed in relation to factors controlling microtubule initiation and distribution.     

Production of specific antibodies to contractile proteins and their use in immunofluorescence microscopy. III. Antiobody against human uterine smooth muscle myosin.

The preparation of highly purified myosin from surgical specimen of human uterine muscle is described. Antibodies were raised in rabbits against this immunogen. In immunodiffusion, they react with uterine and chicken gizzard muscle myosin, no reaction is observed between uterine myosin and the anti-chicken-gizzard- myosin. In immunofluorescence, anti-uterine-myosin stains smooth muscle in the contractile and "modulated" state and non-muscle cells such as fibroblasts, platelets and endothelium of various species. Thus, these antibodies contrast anti-gizzard-myosin, which has previously been shown to be specific for contractile state muscle cells. We therefore conclude that the uterine myosin preparation consists of two immunogens, the one being associated with cell contractility and the other, termed cytoplasmic myosin, with motility and mitosis. The latter is indistinguishable from the myosin present in non-muscle cells and can be absorbed specifically with actomyosin from blood platelets.     

Shaping up for shipping out: PLCgamma signaling of morphology changes in EGF-stimulated fibroblast migration

For effective migration, cells must establish an asymmetry in cell/substratum biophysical interactions permitting cellular protrusive and contractile motive forces to produce net cell body translocation; often this is superficially manifested as a polarized cell shape. This change is most easily noted for epithelial cells, which typically undergo a mesenchymal transition prior to rapid motility, and for hematopoietic cells, which must transition from non-adherent to adherent states. These two situations entail dramatic changes that also involve cell-cell contact and differentiation-related changes, and thus introduce confounding events and signals in defining control elements. Hence, a simpler biochemical and biophysical model system may be useful for gaining fundamental insights into the underlying mechanisms. Fortunately, even relatively "uniform" fibroblasts also undergo an initial shape change to commence locomotion. Investigators have recently begun to probe underlying signals that contribute to the reorganization of the actin cytoskeleton. We describe here a model for fibroblast shape changes involved in epidermal growth factor (EGF) stimulation of motility, focusing on signals through EGF receptor (EGFR) -mediated pathways influencing cytoskeletal organization and cell/substratum adhesion. We present new data addressing specifically phospholipase C-gamma (PLCgamma) pathway activation of actin-modifying proteins, including gelsolin, that contributes to these changes and promotes cell migration by increasing the fraction of cells in a motility-permissive morphology and the time spent in such a state.     

Motile iridophores of a freshwater goby,Odontobutis obscura

Summary Reflecting chromatophores in the dermis of the skin of a freshwater goby, Odontobutis obscura, are of an iridophore type. These chromatophores contain numerous reflecting platelets, which are similar to those in iridophores of other fish and amphibian species. It was found that these iridophores are motile, i.e., these cells respond to certain stimuli with translocation of the platelets within the cells. K⁺ ions induced dispersion of the platelets in excised scale preparations, but not in excised scales from chemically denervated fish. Norepinephrine and melatonin also induced dispersion of the platelets. Alpha-MSH was effective in aggregating these organelles into the centrospheres of the cells. The conclusions reached are: (1) iridophores of O. obscura are motile; (2) the movement of the iridophores is under nervous and hormonal control.     

Electron microscopy of two types of reflecting chromatophores (iridophores and leucophores) in the guppy,Lebistes reticulatus Peters

Summary Reflecting chromatophores in the integument of the guppy, Lebistes reticulatus Peters, are of two distinct types, iridophores and leucophores. The iridophores are smaller and fixed, producing a metallic iridescent color. The cytoplasmic organelles involved in the coloration of iridophores are the reflecting platelets, as in the iridophores of other fish and amphibian species on which earlier reports have been made. Spherical granules of pleiomorphic internal structure, quite variable in size but generally 0.2 m to 1.0 m in diameter, are also numerous in the iridophores. The nature of these granules remains unknown.The leucophores are larger, and highly dendritic; their pigment granules are migratory and they exhibit a dull whitish color. Pigment granules of the leucophores are spherical in form, varying from 0.5–0.8 m in diameter, with a double membrane enclosing the internal fibrous materials. Melamine-treatment of the fish caused degenerative changes in the pigment granules and also the other cytoplasmic organelles of the leucophores, whereas the other kinds of chromatophores, including the iridiophores, remained intact. Some problems in general characterization and classification between these two types of chromatophores were discussed.The author wishes to thank Mr. Yoshiro Yamazaki for his assistance in operating the electron microscope, and Dr. Takao Kajishima (Biological Institute, Nagoya University) for his encouragements     

Ultrastructure of the dermal chromatophores in a lizard (Scincidae: Plestiodon latiscutatus) with conspicuous body and tail coloration

Microscopic observation of the skin of Plestiodon lizards, which have body stripes and blue tail coloration, identified epidermal melanophores and three types of dermal chromatophores: xanthophores, iridophores, and melanophores. There was a vertical combination of these pigment cells, with xanthophores in the uppermost layer, iridophores in the intermediate layer, and melanophores in the basal layer, which varied according to the skin coloration. Skin with yellowish-white or brown coloration had an identical vertical order of xanthophores, iridophores, and melanophores, but yellowish-white skin had a thicker layer of iridophores and a thinner layer of melanophores than did brown skin. The thickness of the iridophore layer was proportional to the number of reflecting platelets within each iridophore. Skin showing green coloration also had three layers of dermal chromatophores, but the vertical order of xanthophores and iridophores was frequently reversed. Skin showing blue color had iridophores above the melanophores. In addition, the thickness of reflecting platelets in the blue tail was less than in yellowish-white or brown areas of the body. Skin with black coloration had only melanophores.     

Hormone induced chromatophore changes in the European tree frog, Hyla arborea, in vitro

The colours of the European tree frog, Hvlu urhorea , depend on three types of chromatophores: in dermo-epidermal direction melanophores, iridophores, and xanthophores. The ability ofthis species to assume a wide range ofcolours implies that very extensive changes in the chromatophores take place, which in turn require control by several regulating factors. The responses of the different chromatophore types to hormones with known melanophore-affecting abilities (α-MSH, β-MSH, ACTH, melatonin) were tested in an in vitro system (freshly explanted skin) using reflectance microspectrophotometry, light microscopy and time-lapse cinemicrography.
α-MSH, β-MSH and ACTH all induce a rapid dispersion of melanosomes during the 10 min after addition. The degree of pigment dispersion induced by ACTH is slightly less than after stimulation with α-MSH or β-MSH.
The iridophores react to MSH or ACTH treatment with a contraction of the entire cell (causing a reduction in reflecting area), and a change in orientation of the platelets, causing a decrease in selective reflectance. The iridophores appear to be especially sensitive to ACTH. A very striking feature of the iridophores when studied with time-lapse cinematography is their strong pulsations (approx. once per minute).
The xanthophores react to MSH and ACTH with a contraction. These cells appear to be sensitive to β-MSH in particular.
Melatonin strongly counteracts the effects of α-MSH, β-MSH and ACTH on all chromatophores.
These studies confirm the dynamic nature not only of the melanophores, but also of the iridophores and xanthophores, as pointed out by Schmidt (1920) and Nielsen (1978a). Furthermore the differences in the time course of the stimulation of the different types of chromatophores by various hormones may provide an experimental basis for the explanation of colour changes in Hyfa arboreu.     

Changes in the ventral dermis and development of iridophores in the anadromous sea lamprey, Petromyzon marinus, during metamorphosis: an ultrastructural study.

The ultrastructural changes that take place in the ventral dermis along with the development of iridophores were examined in the anadromous sea lamprey, Petromyzon marinus, during metamorphosis. There is a disruption of all components of the ventral dermis and a reformation that results in a structure very similar to that prior to metamorphosis. Although not a dermal component, a layer of iridophores develops directly beneath the dermis during late metamorphosis. The dermal endothelium is lost by mid metamorphosis (stage 4) and the highly organized collagenous lamellae making up the bulk of the dermis become disrupted by the migration of fibroblasts into the region. Many of these fibroblasts are involved in the degradation of the lamellae. By stage 5 of metamorphosis some fibroblasts become highly active collagen synthesizing cuboidal shaped cells that align to form a layer above the reformed dermal endothelium. New lamellae are formed by these cuboidal cells which then divide and migrate into the lamellae where they assume the characteristic attenuated appearance of fibroblasts in the adult dermal lamellae region. Iridophores first appear during stage 5 directly beneath the dermal endothelium. Reflecting platelets develop from double membraned vesicles associated with the Golgi apparatus. By late metamorphosis, stacks of trapezoidal shaped platelets fill the cytoplasm of the iridophores. The significance of the changes in the dermis during metamorphosis are discussed. This work is part of a continuing series of studies on the connective tissues in the anadromous sea lamprey.     

LAMELLAE IN THE SPINDLE OF MITOTIC CELLS OF WALKER 256 CARCINOMA

In mitotic cells of Walker 256 carcinoma some four-layered lamellar structures were observed which had the appearance of paired cysternae of the ER. Two inner membranes were regular, smooth surfaced, and closely applied to each other. The two outer membranes were somewhat irregularly placed in relation to the inner pair; they showed attached RNP particles and connections with cysternae of the ER. The membranes often appeared to radiate from the region of the centrosphere towards the compact mass of chromosomes. Thus, they lay amid the spindle fibres and are referred to as "spindle lamellae." They approached the centrioles closely but were not observed to be continuous with them. They appeared to terminate in the pole of the spindle by joining smooth surfaced membranes in the centrosphere. Their equatorial termination was in relation to the chromosomes. At the surface of the chromosome mass they frequently split into two double membranes, which were closely applied to the chromosome substance. The most prominent and complicated membranes were seen in anaphase cells. An hypothesis is advanced which ascribes the development of the nuclear membrane to the spindle lamellae.     

Adaptations of the reed frog Hyperolius viridiflavus (Amphibia,Anura, Hyperoliidae) to its arid environment

Summary Hyperolius viridiflavus nitidulus inhabits parts of the seasonally very hot and dry West African savanna. During the long lasting dry season, the small frog is sitting unhidden on mostly dry plants and has to deal with high solar radiation load (SRL), evaporative water loss (EWL) and small energy reserves. It seems to be very badly equipped to survive such harsh climatic conditions (unfavorable surface to volume ratio, very limited capacity to store energy and water). Therefore, it must have developed extraordinary efficient mechanisms to solve the mentioned problems. Some of these mechanisms are to be looked for within the skin of the animal (e.g. protection against fast desiccation, deleterious effects of UV radiation and overheating). The morphology of the wet season skin is, in most aspects, that of a normal anuran skin. It differs in the organization of the processes of the melanophores and in the arrangement of the chromatophores in the stratum spongiosum, forming no Dermal Chromatophore Unit. During the adaptation to dry season conditions the number of iridophores in dorsal and ventral skin is increased 4–6 times compared to wet season skin. This increase is accompanied by a very conspicuous change of the wet season color pattern. Now, at air temperatures below 35° C the color becomes brownish white or grey and changes to a brilliant white at air temperatures near and over 40° C. Thus, in dry season state the frog retains its ability for rapid color change. In wet season state the platelets of the iridophores are irregularly distributed. In dry season state many platelets become arranged almost parallel to the surface. These purine crystals probably act as quarter-wave-length interference reflectors, reducing SRL by reflecting a considerable amount of the radiated energy input.EWL is as low as that of much larger xeric reptilians. The impermeability of the skin seems to be the result of several mechanisms (ground substance, iridophores, lipids, mucus) supplementing each other.The light red skin at the pelvic region and inner sides of the limbs is specialized for rapid uptake of water allowing the frog to replenish the unavoidable EWL by using single drops of dew or rain, available for only very short periods.     

Malleable skin coloration in cephalopods: selective reflectance, transmission and absorbance of light by chromatophores and iridophores

Nature's best-known example of colorful, changeable, and diverse skin patterning is found in cephalopods. Color and pattern changes in squid skin are mediated by the action of thousands of pigmented chromatophore organs in combination with subjacent light-reflecting iridophore cells. Chromatophores (brown, red, yellow pigment) are innervated directly by the brain and can quickly expand and retract over underlying iridophore cells (red, orange, yellow, green, blue iridescence). Here, we present the first spectral account of the colors that are produced by the interaction between chromatophores and iridophores in squid (Loligo pealeii). Using a spectrometer, we have acquired highly focused reflectance measurements of chromatophores, iridophores, and the quality and quantity of light reflected when both interact. Results indicate that the light reflected from iridophores can be filtered by the chromatophores, enhancing their appearance. We have also measured polarization aspects of iridophores and chromatophores and show that, whereas structurally reflecting iridophores polarize light at certain angles, pigmentary chromatophores do not. We have further measured the reflectance change that iridophores undergo during physiological activity, from "off" to various degrees of "on", revealing specifically the way that colors shift from the longer end (infra-red and red) to the shorter (blue) end of the spectrum. By demonstrating that three color classes of pigments, combined with a single type of reflective cell, produce colors that envelop the whole of the visible spectrum, this study provides an insight into the optical mechanisms employed by the elaborate skin of cephalopods to give the extreme diversity that enables their dynamic camouflage and signaling.     

Unusual development of light-reflecting pigment cells in intact and regenerating tail in the periodic albino mutant of Xenopus laevis

Unusual light-reflecting pigment cells, “white pigment cells”, specifically appear in the periodic albino mutant (a ^p /a ^p ) of Xenopus laevis and localize in the same place where melanophores normally differentiate in the wild-type. The mechanism responsible for the development of unusual pigment cells is unclear. In this study, white pigment cells in the periodic albino were compared with melanophores in the wild-type, using a cell culture system and a tail-regenerating system. Observations of both intact and cultured cells demonstrate that white pigment cells are unique in (1) showing characteristics of melanophore precursors at various stages of development, (2) accumulating reflecting platelets characteristic of iridophores, and (3) exhibiting pigment dispersion in response to α-melanocyte stimulating hormone (α-MSH) in the same way that melanophores do. When a tadpole tail is amputated, a functionally competent new tail is regenerated. White pigment cells appear in the mutant regenerating tail, whereas melanophores differentiate in the wild-type regenerating tail. White pigment cells in the mutant regenerating tail are essentially similar to melanophores in the wild-type regenerating tail with respect to their localization, number, and response to α-MSH. In addition to white pigment cells, iridophores which are never present in the intact tadpole tail appear specifically in the somites near the amputation level in the mutant regenerating tail. Iridophores are distinct from white pigment cells in size, shape, blue light-induced fluorescence, and response to α-MSH. These findings strongly suggest that white pigment cells in the mutant arise from melanophore precursors and accumulate reflecting platelets characteristic of iridophores.     

Distribution of multiple centrospheres determines migration of BHK syncitia

After fusion of BHK cells with polyethylene glycol, the resulting syncitia contained in 77% of the cases multiple microtubule organizing centers (MTOCs), which were aggregated into a common centrosphere. Based on the observation of phagokinetic tracks, we found that the syncitia were able to locomote if the MTOCs aggregated into a common centrosphere cluster, and the clustered centrospheres were excluded from the cluster of nuclei of the syncitium. The results suggest that each individual pair of one nucleus and one centrosphere contributes, in a process of vectorial addition, its individual polarity to the polarity of the syncitium. Thus the widely accepted idea that the centrosphere is involved in the determination of cell polarity can be generalized beyond the case of single cells.     

Production of specific antibodies to contractile proteins and their use in immunofluorescence microscopy

Summary The preparation of highly purified myosin from surgical specimen of human uterine muscle is described. Antibodies were raised in rabbits against this immunogen. In immunodiffusion, they react with uterine and chicken gizzard muscle myosin, no reaction is observed between uterine myosin and the anti-chicken-gizzard- myosin. In immunofluorescence, antiuterine-myosin stains smooth muscle in the contractile and modulated state and non-muscle cells such as fibroblasts, platelets and endothelium of various species. Thus, these antibodies contrast anti-gizzard-myosin, which has previously been shown to be specific for contractile state muscle cells. We therefore conclude that the uterine myosin preparation consists of two immunogens, the one being associated with cell contractility and the other, termed cytoplasmic myosin, with motility and mitosis. The latter is indistinguishable from the myosin present in non-muscle cells and can be absorbed specifically with actomyosin from blood platelets.Abbreviations ATP Adenosine triphosphate - DNAse I Deoxyribonuclease I - DTE Dithioerythritol - SDS Sodiumdodecylsulfate - PAGE Polyacrylamide electrophoresis