Zebrafish endzone regulates neural crest-derived chromatophore differentiation and morphology

The development of neural crest-derived pigment cells has been studied extensively as a model for cellular differentiation, disease and environmental adaptation. Neural crest-derived chromatophores in the zebrafish (Danio rerio) consist of three types: melanophores, xanthophores and iridiphores. We have identified the zebrafish mutant endzone (enz), that was isolated in a screen for mutants with neural crest development phenotypes, based on an abnormal melanophore pattern. We have found that although wild-type numbers of chromatophore precursors are generated in the first day of development and migrate normally in enz mutants, the numbers of all three chromatophore cell types that ultimately develop are reduced. Further, differentiated melanophores and xanthophores subsequently lose dendricity, and iridiphores are reduced in size. We demonstrate that enz function is required cell autonomously by melanophores and that the enz locus is located on chromosome 7. In addition, zebrafish enz appears to selectively regulate chromatophore development within the neural crest lineage since all other major derivatives develop normally. Our results suggest that enz is required relatively late in the development of all three embryonic chromatophore types and is normally necessary for terminal differentiation and the maintenance of cell size and morphology. Thus, although developmental regulation of different chromatophore sublineages in zebrafish is in part genetically distinct, enz provides an example of a common regulator of neural crest-derived chromatophore differentiation and morphology.     

Chromatophore radial muscle fibers anchor in flexible squid skin

Cephalopod skin is soft, flexible, and produces rapid color changes for camouflage and signaling primarily by regulating the shapes of its numerous chromatophore organs. Each chromatophore has 10–30 radial muscle cells, termed fibers, under central nervous system control. Each fiber contains myofilaments that contract in concert to stretch the pigment‐containing cell from its punctate, spherical state to a fully expanded thin disk of color. Expansion occurs in less than one second and can result in a 14‐fold expansion in pigment cell diameter. We investigated the anchoring mechanism of radial muscle fibers that expand pigment cells in the longfin squid, Doryteuthis (Loligo) pealeii. The proximal Active Zone of a radial muscle fiber adheres to the pigment cell within an ensheathing sinus. The distal portion forms terminal arbors, thereby increasing the surface area, to adhere it to the dermal extracellular matrix (ECM). While the muscle fiber is attached to the pigment cell with haptosomes, the remainder of the fiber is adhered to the surrounding basal lamina (part of the ECM) by numerous, closely spaced, small costamere‐like projections. Branching of the radial muscle fiber termini and the costamere‐like attachments are key anatomical specializations that anchor the radial muscle fibers in the pliable skin while allowing the freedom of movement required for large changes in pigment cell diameter. We postulate that these features may be relevant for the development of soft actuation models in materials science.     

The morphology of dermal chromatophores in the infrared-reflecting glass-frog Centrolenella fleischmanni

The morphology and organization of chromatophores in the neotropical glass-frog, Centrolenella fleischmanni (family Centrolenidae), were studied with both light and electron microscopes. Four types of pigment cells are described in the dorsal skin. The fine structure of two chromatophores corresponds to the typical amphibian xanthophore and iridophore; one is similar to the unusual melanophore found in phyllomedusine hylids; the fourth cell type is unlike any chromatophore previously described. Pigment granules in the unusual chromatophore are moderately electron-dense and have an irregular shape, suggesting a fluid composition. This pigment appears to be laid down in organelles similar in appearance to pterinosomes. The organization of pigment cells in this species differs from that of other green, leaf-sitting frogs in that there are few discrete groups resembling “dermal chromatophore units.” It is suggested that the unusual new pigment cell contributes significantly to the overall green color of C. fleischmanni.     

Skin color in the squids Loligo pealii and Loligo opalescens

Summary Rapid, physiological color changes seen in the skin of cephalopods are due to a unique anatomical system composed of chromatophore organs and iridophores. The morphology and ultrastructure of the chromatophores was studied in the squids Loligo pealii Lesueur and Loligo opalescens Berry. A three-dimensional model of a brown chromatophore was reconstructed from serial sections for the electron microscope.The chromatophore organ is composed of a central nucleated pigment cell, 10–30 obliquely striated muscle cells (radially arranged on the equator of the pigment cell), axons, Schwann cells, and sheath cells. The pigment cell consists of a central aggregation of pigment granules and surrounding peripheral cytoplasmic compartments. These regions are incompletely separated by an electron-dense, sac-like structure, the pigment container. Proximal portions of a muscle cell contact the pigment cell in regions called myo-chromatophore junctions. Neuromuscular and myo-muscular junctions are also present.The results presented are discussed in terms of previous morphological and physiological studies of chromatophores.Part of a study submitted in partial fulfillment of the requirement for the degree of Ph. D. (Anatomy), the Graduate School of Basic Medical Sciences, New York Medical College, New York, N.Y. 10029.The research reported here was in part supported by grants from the Health Research Council of the City of New York (U-1008) and United States Public Health Service, General Research Grant No. FR-05398.Report on some of this material was given at the Annual Meeting of the American Association of Anatomists, Philadelphia, Pennsylvania, April 19–22, 1971.     

Chromatophore Organs, Reflector Cells, Iridocytes and Leucophores in Cephalopods

The chromatophore organs of Lohgo are each composed of fivetypes of cells: a central pigment cell: radially arranged, obliquelystriated muscle fibers: neuronal processes; glial cells: andan investment of sheath cells. Sheath cells are absent in Octopuschromatophore organs. The cycle of expansion and retractionof a chromatophore organ may occur within the order of a second.It is clear that the muscle fibers expand the pigment cell andspread out the pigment granules. The pigment is contained withinan unusual, filamentous, cytoplasmic compartment called thecytoelastic sacculus. This compartment has elastic properties. Reflector cells and iridocytes produce structural colors eventhough their components are colorless. Reflector cells in Octopusbear peripheral sets of leaf-like reflecting lamellae calledreflectosomes: these contain proteinaceous platelets with ahigh refractive index (1.42). In each reflectosome the reflectinglamellae are separated by gaps that are about equal to the thicknessof the lamellae, but have a lower refractive index (1.33). Reflectosomesare believed to reflect light and to function as thin-film interferencedevices. Iridocytes in squid and cuttlefish contain iridosomes that arealso composed of sets of ribbon-like platelets but these arelocated centrally within the cell body. The platelets are usuallyoriented on edge with respect to the surface of the skin. Thepossibility that dermal iridocytes may act as diffraction gratingsis discussed. Leucophores have thousands of processes that containglobules of protein with a high refractive index. These cellsscatter light of all wave lengths and appear white in whitelight.     

Molecular and developmental contributions to divergent pigment patterns in marine and freshwater sticklebacks

Pigment pattern variation across species or populations offers a tractable framework in which to investigate the evolution of development. Juvenile threespine sticklebacks (Gasterosteus aculeatus) from marine and freshwater environments exhibit divergent pigment patterns that are associated with ecological differences. Juvenile marine sticklebacks have a silvery appearance, whereas sticklebacks from freshwater environments exhibit a pattern of vertical bars. We investigated both the developmental and molecular basis of this population‐level variation in pigment pattern. Time course imaging during the transition from larval to juvenile stages revealed differences between marine and freshwater fish in spatial patterns of chromatophore differentiation as well as in pigment amount and dispersal. In freshwater fish, melanophores appear primarily within dark bars whereas iridophores appear within light bars. By contrast, in marine fish, these chromatophores are interspersed across the flank. In addition to spatially segregated chromatophore differentiation, pigment amount and dispersal within melanophores varies spatially across the flank of freshwater, but not marine fish. To gain insight into the molecular pathways that underlie the differences in pigment pattern development, we evaluated differential gene expression in the flanks of developing fish using high‐throughput cDNA sequencing (RNA‐seq) and quantitative PCR. We identified several genes that were differentially expressed across dark and light bars of freshwater fish, and between freshwater and marine fish. Together, these experiments begin to shed light on the process of pigment pattern evolution in sticklebacks.     

On the fine structure of the Octopus iris

Summary The Octopus iris is composed of five different layers: A, the external epithelium; B, the chromatophore layer; C, the iridocyte layer; D, the layer of muscles and collagen strands; E, the pigment epithelium. The nerves innervating the sphincter and the chromatophore muscles are identified and their neuromuscular junction is described. The motor endings of chromatophore nerves have an additional ending in presynaptic position which probably functions as a modifier of neuromuscular transmission. The chromatophores are naked and exhibit a tubular channel system between plasmalemma and pigment container which looks similar to the T-system of muscle cells.The financial support of this investigation by the Swiss National Foundation is gratefully acknowledged.     

Cellular Aspects of the Control of Physiological Color Changes in Crustaceans

SYNOPSIS. Red chromatophores(erythrophores) of the prawn, Palaemonetesvulgaris, are controlled by pigment—dispersing and -concentratinghormones. Recent experiments on the modes of action of thesehormones are described, followed by a theory which satisfactorilyexplains the data. Red pigment-concentrating hormone is dependentupon sodium ions for a strong response to occur. There is asimilar dependency of red pigment—dispersing hormone uponcalcium ions. Ouabain inhibits the response to red pigment—concentratinghormone; tetrodotoxin enhances it. Erythrophores with maximallydispersed pigment had a transmembrane potential of 55±15mv inside negative in one series of experiments and 56±4mv in another. No appreciable changes in permeability occurwhen depolarizing and hyperpolarizing currents are passed througha microelectrode within the chromatophore. Red pigmentconcentratinghormone causes hyperpolarization of the transmembrane potential.The magnitude of hyperpolarization is directly related to thedegree of pigment concentration. Adenosine 3`;, 5`-monophosphate(cyclic AMP) causes dispersion of the red pigment but has nopigment-concentrating effect. The primary action of red pigmentconcentratinghormone is most likely stimulation of a pump which exchangessodium ions from inside the chromatophore with potassium ionsfrom the outside, whereas red pigment-dispersing hormone quitelikely stimulates entry of calcium ions into the chromatophore.     

Ultrastructure and Function of Cephalopod Chromatophores

SYNOPSIS. Each chromatophore organ consists of a pigment celland of several radial muscle fibers that represent separatecells. The pigment granules are contained within an elasticsacculus within the pigment cell. The sacculus is attached aroundthe equator of the chromatophore to the cell membrane by zonalhaptosomes. In turn, the cell membrane is attached to the radialmuscle fibers by a dense basal lamina. The cell membrane ofthe retracted chromatophore is highly folded. Contraction ofthe radial muscle fibers is initiated by (a) excitatory junctionpotentials, (b) miniature potentials, or (c) spike potentials.The latter arise spontaneously in the muscle fibers when thesehave undergone some internal (metabolic?) change. The contractionof the muscle fibers causes expansion of the pigment-containingsacculus. Relaxation of the muscle fibers permits the sacculusto assume its original lenticular or near-spherical shape; theenergy for this is stored within theexpanded elastic componentsof the sacculus. In normal skin the chromatophore organs areentirely under the control of the central nervous system, themuscle fibers being activated only by local, excitatory postsynapticpotentials initiated by motor nerve impulses. That postsynapticpotentials are non-propagating insures that individual motorfibers can be activated individually, thus permitting a delicatecontrol of skin color by recruitment as well as by frequency.Tonic contractions and pulsations, involving spontaneous releaseof transmitter from nerve terminals and spike generation withinthe muscle fibers, respectively, are the result of altered,abnormal conditions within the skin.     

Ultrastructure of the chromatophore system on the tracheal bladders of the phantom larva of Chaoborus crystallinus (Insecta,Diptera)

The chromatophore system on the tracheal bladders of the phantom larva of Chaoborus crystallinus has been investigated by light and electron microscopy. The pigment cells are attached to a restricted region of the outer surface of the bladders and have the capacity to change both their shape and position on the bladder in response to changes in background illumination. The whole pigment system is tightly spanned by an extracellular membrane, which is in contact with two small muscles inserting at the anterior inner wall of the bladders.Nachdem das Manuskript eingereicht worden war, ist Walter Weber verstorben. An seiner Stelle hat Herr Dr. Lehmann (Köln) zur Fertigstellung der Druckfassung der Arbeit beigetragen     

Xanthophores in chromatophore groups of the premigratory neural crest initiate the pigment pattern of the axolotl larva

Summary The barred pigment pattern (Lehman 1957) of the axolotl larva is best observed from stage 41 onwards, where it already consists of alternating transverse bands of melanophores and xanthophores along the dorsal side of the trunk. The present study investigateswhen the two populations of neural crest derived chromatophores, melanophores and xanthophores become determined andhow they interact to create the barred pigment pattern. The presence of phenol oxidase (tyrosinase) in melanophores (revealed by dopa incubation) and pteridines in xanthophores (visualized by fluorescence) were used as markers for cell differentiation in order to recognize melanophores and xanthophores before they became externally visible. It was found that melanophores and xanthophores were already determined in the premigratory neural crest, at stages 30/31 and 35–36, respectively. Between stages 35–36 and 38 they were arranged in a prepattern of several distinct, mixed chromatophore groups along the dorsal trunk, morphologically correlated in the scanning electron microscope with humps on the original crest cell string. While the occurrence of xanthophores was restricted to the chromatophore groups and around them, melanophores were already uniformly distributed in the dorsolateral flank area, having migrated from trunk neural crest portions including the groups. The bar component of the pigment pattern was subsequently initiated by xanthophores, which caused melanophores in and around the chromatophore groups to fade or become invisible. The barred pattern was established by the formation of alternating clusters of like cells, melanophores and xanthophores.     

Pigment Cell Refugia in Homeotherms—The Unique Evolutionary Position of the Iris

Homeotherms are generally considered to lack classical active dermal pigment cells (chromatophores) in their integument, attributable to the development of an outer covering coat of hair or feathers. However, bright colored dermal pigment cells, comparable to chromatophores of lower vertebrates, are found in the irides of many birds. We propose that, because of its exposed location, the iris is an area in which color from pigment cells has sustained a selective advantage and appears to have evolved independently of the general integument. In birds, the iris appears to have retained the potential for the complete expression of all dermal chromatophore types. Differences in cell morphology and the presence of unusual pigments in birds are suggested to be the result of evolutionary changes that followed the divergence of birds from reptiles. By comparison, mammals appear to have lost the potential for producing iridophores, xanthophores, or erythrophores comparable to those of lower vertebrates, even though some species possess brightly colored irides. It is proposed that at least one species of mammal (the domestic cat) has recruited a novel iridial reflecting pigment organelle originally developed in the choroidal tapetum lucidum. The potential presence of classical chromatophores in mammals remains open, as few species with bright irides have been examined.     

Pigment cell refugia in homeotherms--the unique evolutionary position of the iris.

Homeotherms are generally considered to lack classical active dermal pigment cells (chromatophores) in their integument, attributable to the development of an outer covering coat of hair or feathers. However, bright colored dermal pigment cells, comparable to chromatophores of lower vertebrates, are found in the irides of many birds. We propose that, because of its exposed location, the iris is an area in which color from pigment cells has sustained a selective advantage and appears to have evolved independently of the general integument. In birds, the iris appears to have retained the potential for the complete expression of all dermal chromatophore types. Differences in cell morphology and the presence of unusual pigments in birds are suggested to be the result of evolutionary changes that followed the divergence of birds from reptiles. By comparison, mammals appear to have lost the potential for producing iridophores, xanthophores, or erythrophores comparable to those of lower vertebrates, even though some species possess brightly colored irides. It is proposed that at least one species of mammal (the domestic cat) has recruited a novel iridial reflecting pigment organelle originally developed in the choroidal tapetum lucidum. The potential presence of classical chromatophores in mammals remains open, as few species with bright irides have been examined.     

Kinetics and yields of bacteriochlorophyll fluorescence: redox and conformation changes in reaction center of <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

Induction of the bacteriochlorophyll fluorescence under rectangular shape of intense laser diode illumination (1 W cm(-2), 808 nm) was measured over wide time range from 10 mus to 4 s in whole cells, chromatophore and isolated reaction center protein of wild type and carotenoid-less mutant (R-26.1) of purple photosynthetic bacterium Rhodobacter sphaeroides. While the antenna-containing species showed large and positive variable fluorescence (F (v)) to initial fluorescence (F (0)) (F (v)/F (0) approximately 4.5 in whole cells), the isolated RC had negative change (F (v)/F (0) approximately -0.6) during photochemistry. In chromatophore from R-26.1, only seven times higher rate was measured than in isolated reaction center under identical experimental conditions. The enhancement effect of large antenna on the rate of photochemistry in chromatophore was partially compensated by the favorable pigment absorption properties in isolated RC. The transition from membrane bound to isolated form of the reaction center was probed by titration of zwitterionic detergent LDAO in chromatophore, and at 0.03% LDAO concentration, sharp change of the variable fluorescence was observed. The sudden drop was explained by the formation of LDAO micelles. After the photochemical phase, additional change of fluorescence yield could be observed in isolated RC considered as manifestation of long-living conformations of the trapped redox states of the protein characterized by non-exponential kinetics. Strong support was provided for use of the fluorescence induction to track structural and conformation changes at their earliest phases in chromatophores and isolated reaction centers.     

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.     

Properties of a pigment system consisting of carotenoids and reaction centre pigments in chromatophores of Rhodospirillum rubrum

A pigment system containing carotenoids and oxidised reaction centre pigments is present in chromatophores of Rhodospirillum rubrum and this pigment system may cause fluorescence quenching when a still unidentified chromatophore component is in its oxidised state. Besides by its action spectrum, this pigment system is characterised by the time course and level of light saturation of the effect. The quenching of bacteriochlorophyll fluorescence is abolished when the permeability of the chromatophore membranes is affected. The quenching effect is correlated with a reversible absorption decrease of B 880. A possible function for this pigment system is discussed.     

The Physiology and Pharmacology of Crustacean Chromatophores

The color change system of crustaceans is being investigatedalong a broad front. The techniques being used include physiological,pharmacological, biochemical, immunocytochemical, and ultrastructuralones. The problems investigators are seeking answers to includethe cellular bases for pigment granule translocation, the numberand specificity of the chromatophorotropic hormones responsiblefor the color changes, and the identity of the neuroregulators(neurotransmitters and neuromodulators) that control the releaseof these hormones. With respect to the cellular bases of pigmentgranule translocation, microtubules and a microtrabecular latticeare prime candidates as organelles that might be responsiblefor the pigment granule movements. Pigment dispersing and pigmentconcentrating neuropeptides have been identified. Some exhibitno specificity with respect to the chromatophore type they activate.Others show high specificity, affecting only one specific typeof chromatophore, such as the melanophore. Several putativeneuroregulator candidates have been identified as possibly havinga role in controlling chromatophorotropic hormone release. Theseinclude 5-hydroxytryptamine, dopamine, norepinephrine, octopamine,and gamma-aminobutyric acid. Some, like the first three, stimulatehormone release, whereas the latter two have inhibitory actions.     

Possible Involvement of Cone Opsins in Distinct Photoresponses of Intrinsically Photosensitive Dermal Chromatophores in Tilapia Oreochromis niloticus

Dermal specialized pigment cells (chromatophores) are thought to be one type of extraretinal photoreceptors responsible for a wide variety of sensory tasks, including adjusting body coloration. Unlike the well-studied image-forming function in retinal photoreceptors, direct evidence characterizing the mechanism of chromatophore photoresponses is less understood, particularly at the molecular and cellular levels. In the present study, cone opsin expression was detected in tilapia caudal fin where photosensitive chromatophores exist. Single-cell RT-PCR revealed co-existence of different cone opsins within melanophores and erythrophores. By stimulating cells with six wavelengths ranging from 380 to 580 nm, we found melanophores and erythrophores showed distinct photoresponses. After exposed to light, regardless of wavelength presentation, melanophores dispersed and maintained cell shape in an expansion stage by shuttling pigment granules. Conversely, erythrophores aggregated or dispersed pigment granules when exposed to short- or middle/long-wavelength light, respectively. These results suggest that diverse molecular mechanisms and light-detecting strategies may be employed by different types of tilapia chromatophores, which are instrumental in pigment pattern formation.     

Color change in exercising crabs: evidence for a hormone

Summary Three species of crabs exercised to fatigue showed a blanching and/or reddening of the body and legs. InUca pugilator this effect was due to white and red pigment dispersion in the leucophores and erythrophores, respectively, and a black pigment concentration in the melanophores. The pigment movements were induced by factor(s) present in the blood of exercisingUca; blood (hemolymph) removed from an exercised crab and injected into the isolated leg segment of another individual cause pigment movements similar to those seen in intact fatigued crabs. The blood of exercisedUca also caused similar chromatophore changes in isolated leg segments of the crabSesarma cinereum. The evidence suggests that blood-borne factor(s) related or identical to chromatophorotropins are released during vigorous exercise in crabs. We speculate that the effects of these exercise factor(s) are secondary to possible effects on carbohydrate and lipid metabolism associated with exercise.     

Teilungsverhalten der Chromatophoren in bezug auf die Mitose während des Lebenszyklus vonDiatoma hiemale var.mesodon

The vegetative life cycle ofDiatoma hiemale var.mesodon (Ehr.)Grun. living in a spring has been studied under natural conditions. In the beginning the cells have a constant number of 8 chromatophores which are divided into 16 during cell growth. Chloroplast division is finished before nuclear division starts. The young daughter cells have again 8 chromatophores. In the course of cell division a plastic remodelling of the chromatophores and a simplifying of their shape occurs. Besides single cells also populations have been studied to follow the temporal progress of chromatophore division, mitosis and cell growth. The results are evaluated by indices and demonstrated by a diagram. The maximum of chromatophore divisions preceds the maximum of mitoses by several hours, while the cell growth is in correlation with the chromatophore division. Minima of the other parameters were found before mitosis is starting and after it is finished. Our results are discussed with regard to the semiautonomy of the plastids. From the morphological point of view this concept is supported by the mode of division and by the anticipation of the chromatophore division. The number of chromatophores at the beginning (8) and at the end (16) of the life cycle is constant. The life cycle is classified into stages of cell growth, chromatophore division, stagnation, mitosis and differentiation of the daughter cells.