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.     

Invertebrate neuropeptide hormones

The development of a long-term research program on the neurosecretory hormones of arthropods is described. The purification and full characterization of the first invertebrate neurohormones, the red pigment-concentrating hormone (RPCH) and the distal retinal pigment hormone (DRPH) demonstrated that they are peptides, an octapeptide and an octadecapeptide, respectively. Physiological function studies with the pure hormones and their synthetic preparations showed that the RPCH acts as a general pigment-concentrating hormone (PCH), and that the DRPH, in addition to its light-adaptive function, also constitutes a general pigment-dispersing hormone (PDH). In the regulation of the color-adaptation of the animals, the two hormones act as antagonists. The chromatophorotropic activities are widely distributed within the arthropod neuroendocrine systems. Purification of the pigment-concentrating activities from the locust corpora cardiaca lead to the isolation and characterization of the first insect neurohormones, the adipokinetic hormones (AKH I and AKH II). These two hormones, AKH I being a decapeptide and AKH II being an octapeptide, are close structural analogs to the crustacean PCH, demonstrating a common evolution of arthropod neurohormones. The hormones of this PCH-family all cross-react, but structure-function studies of the hormones show that quite different parts of their structure are involved in their binding to the various receptors.     

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.     

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.     

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.     

Esrom, an ortholog of PAM (protein associated with c-myc), regulates pteridine synthesis in the zebrafish

Zebrafish esrom mutants have an unusual combination of phenotypes: in addition to a defect in the projection of retinal axons, they have reduced yellow pigmentation. Here, we investigate the pigment phenotype and, from this, provide evidence for an unexpected defect in retinal neurons. Esrom is not required for the differentiation of neural crest precursors into pigment cells, nor is it essential for cell migration, pigment granule biogenesis, or translocation. Instead, loss of yellow color is caused by a deficiency of sepiapterin, a yellow pteridine. The level of several other pteridines is also affected in mutants. Importantly, the cofactor tetrahydrobiopterin (BH4) is drastically reduced in esrom mutants. Mutant retinal neurons also appear deficient in this pteridine. BH4-synthesizing enzymes are active in mutants, indicating a defect in the regulation rather than production of enzymes. Esrom has recently been identified as an ortholog of PAM (protein associated with c-myc), a very large protein involved in synaptogenesis in Drosophila and C. elegans. These data thus introduce a new regulator of pteridine synthesis in a vertebrate and establish a function for the Esrom protein family outside synaptogenesis. They also raise the possibility that neuronal defects are due in part to an abnormality in pteridine synthesis.     

Endocrine Mechanisms in Crayfish, with Emphasis on Reproduction and Neurotransmitter Regulation of Hormone Release

SYNOPSIS. The neuroendocrine system of crustaceans, crayfishin particular, has been extensively studied. As among vertebrates,a wide variety of processes are hormonally regulated, includingreproduction, growth, carbohydrate metabolism and color changes.The sinus gland, a neurohemal organ in the crayfish eyestalk,is the major neuroendocrine center. Herein, crayfish reproductivecycles and their hormonal control, particularly by the gonad-stimulatingand gonad/inhibiting hormones, are a major focus. In addition,the aminergic and peptidergic neuroregulators identified incrayfish central nervous organs that either have been or likelymay be shown to have neurotransmitter/neuromodulator roles incontrolling hormone release are discussed with respect to localizationand identified roles.     

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.     

Fish chromatophores as cytosensors in a microscale device: detection of environmental toxins and bacterial pathogens

Fish chromatophores from Betta splendens are used as the cytosensor element in the development of a portable microscale device capable of detecting certain environmental toxins and bacterial pathogens by monitoring changes in pigment granule distribution. The adaptation of chromatophores to a microscale environment has required the development of enabling technologies to produce miniaturized culture chambers, to integrate microfluidics for sample delivery, to miniaturize image capture, and to design new statistical methods for image analyses. Betta splendens chromatophores were selected as the cytosensor element because of their moderate size, their toleration of close contact, and most importantly, for their responses to a broad range of chemicals and pathogenic bacteria. A miniaturized culture chamber has been designed that supports chromatophore viability for as long as 3 months, and that can be easily transported without damage to the cells. New statistical methods for image analyses have been developed that increase sensitivity and also decrease the time required for detection of significant changes in pigment granule distribution. Betta chromatophores have been tested for their responses to selected pathogenic bacteria and chemical agents. We discuss in detail the aggregation of pigment granules seen when chromatophores are incubated with Bacillus cereus, a common cause of food poisoning. Also described are the more subtle responses of chromatophores to a class of environmental chemical toxins, polynuclear aromatic hydrocarbons. We show that the chromatophores are able to detect the presence of certain polynuclear aromatic hydrocarbons at concentrations lower than the Environment Protection Agency (EPA) 550.1 standards.     

Clonal heterogeneity in physiological properties of melanized cells induced from goldfish erythrophoroma cell lines

Abstract. Cells of the uncloned goldfish erythrophoroma lines, GEM-81 and GEM-218, have been induced to melanize by cultivation in autologous serum. The melanized cells continue to proliferate and exhibit clonal heterogeneity in terms of morphology, growth rates, contact behavior and pigment content, and distribution and translocation in response to hormones. Based on these characteristics and those of their normal counterparts, the melanized tumor cells have been categorized as type-I and type-I1 melanocytomas, and melanophoromas. The melanophoroma cells are capable of pigment translocation in response to epinephrine, melatonin, and/or MSH, whereas melanocytoma cells are not. The distinguishing characteristics of each type are apparent at the first appearance of melanized cells and appear to be stable except in some type-II melanocytoma clones which contain cells capable of differentiating into melanophoroma cells in long-term cultures. It appears that the parent erythrophoroma lines contain stem cells, melanoblastomas, which are capable of melanogenesis. These stem cells may themselves be a heterogeneous population with respect to the characteristics of the melanized cells to which they give rise.     

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.     

Penetration of protein toxins into cells

AB toxins deliver their enzymatically active A domain to the cytosol. Some AB-toxins are able to penetrate cellular membranes from endosomes where the low pH triggers their translocation. One such toxin is diphtheria toxin and important features of its translocation mechanism have been unraveled during the last year. Other toxins depend on retrograde transport through the secretory pathway to the ER before translocation, and recent findings suggest that these toxins take advantage of the ER translocation machinery normally used for transport of cellular proteins. In addition, the intracellular targets of many of these toxins have been identified recently.     

THE DEVELOPMENT OF PIGMENT GRANULES IN THE EYES OF WILD TYPE AND MUTANT DROSOPHILA MELANOGASTER

The eye pigment system in Drosophila melanogaster has been studied with the electron microscope. Details in the development of pigment granules in wild type flies and in three eye color mutants are described. Four different types of pigment granules have been found. Type I granules, which carry ommochrome pigment and occur in both primary and secondary pigment cells of ommatidia, are believed to develop as vesicular secretions by way of the Golgi apparatus. The formation of Type II granules, which are restricted to the secondary pigment cells and contain drosopterin pigments, involves accumulation of 60- to 80-A fibers producing an elliptical granule. Type III granules appear to be empty vesicles, except for small marginal areas of dense material; they are thought to be abnormal entities containing ommochrome pigment. Type IV granules are characteristic of colorless mutants regardless of genotype, and during the course of development they often contain glycogen, ribosomes, and show acid phosphatase activity; for these reasons and because of their bizarre and variable morphology, they are considered to be autophagic vacuoles. The 300-A particles commonly found in pigment cells are identified as glycogen on the basis of their morphology and their sensitivity to salivary digestion.     

Eye color pigment granules in Drosophila mauritiana: mosaics produced by excision of a transposable element

Compound eyes of the white-peach (wpch) mutant strain of Drosophila mauritiana have some pigment and receptor cells with wild-type eye color pigmentation. These eyes are mosaic, because excision of a transposable element reverts wpch to wild type during the development of somatic cells. Wild-type patches have three types of pigment granule residing in three respective cell types: primary pigment cells, secondary pigment cells, and retinula (visual receptor) cells. Most aspects of these granules, as well as all other aspects of compound eye ultrastructure, are exactly as in the better studied sibling species D. melanogaster. In the wpch parts of the eye, small and giant unpigmented "pigment granules" reside in secondary pigment cells. These white granules are just like the corresponding granules of w mutant D. melanogaster. Small vs. large patches of pigmented cells likely represent excision events occurring late vs. early respectively during development. Mosaics of eye color markers have been important in developmental analyses; the ease of constructing mosaics of D. mauritiana gives this preparation advantages for mosaic analyses.     

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.     

Vertebrate melanophores as potential model for drug discovery and development: A review

Drug discovery in skin pharmacotherapy is an enormous, continually expanding field. Researchers are developing novel and sensitive pharmaceutical products and drugs that target specific receptors to elicit concerted and appropriate responses. The pigment-bearing cells called melanophores have a significant contribution to make in this field. Melanophores, which contain the dark brown or black pigment melanin, constitute an important class of chromatophores. They are highly specialized in the bidirectional and coordinated translocation of pigment granules when given an appropriate stimulus. The pigment granules can be stimulated to undergo rapid dispersion throughout the melanophores, making the cell appear dark, or to aggregate at the center, making the cell appear light. The major signals involved in pigment transport within the melanophores are dependent on a special class of cell surface receptors called G-protein-coupled receptors (GPCRs). Many of these receptors of adrenaline, acetylcholine, histamine, serotonin, endothelin and melatonin have been found on melanophores. They are believed to have clinical relevance to skin-related ailments and therefore have become targets for high throughput screening projects. The selective screening of these receptors requires the recognition of particular ligands, agonists and antagonists and the characterization of their effects on pigment motility within the cells. The mechanism of skin pigmentation is incredibly intricate, but it would be a considerable step forward to unravel its underlying physiological mechanism. This would provide an experimental basis for new pharmacotherapies for dermatological anomalies. The discernible stimuli that can trigger a variety of intracellular signals affecting pigment granule movement primarily include neurotransmitters and hormones. This review focuses on the role of the hormone and neurotransmitter signals involved in pigment movement in terms of the pharmacology of the specific receptors.     

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.     

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.     

Chemical Properties and Physiological Actions of Crustacean Chromatophorotropins

The crustacean pigment-translocating hormones, the red pigment-concentratinghormone (RPCH), an octapeptide, and the light-adapting distalretinal pigment hormone (DRPH), an octadecapeptide, are thefirst invertebrate neurohormones to be fully characterized.Studies with both purified and synthetic hormones show that,in certain decapods, RPCH is a general pigment-concentratinghormone (PCH), affecting the pigments of all kinds of chromatophores(erythrophores, xanthophores, leucophores and melanophores);the DRPH seems to serve not only light-adapting function, butalso act as a general chromatophore pigment-dispersing hormone(PDH). The two hormones thus function as antagonists when regulatingthe color-adaptation of the decapod crustaceans. PCH activityis widely distributed within the arthropod endocrine systems.The first characterized insect neurohormones, the locust adipokinetichormones (AKH I and AKH II), show close structural similaritiesto the crustacean hormone, indicating a common evolution ofsome of the arthropod neurohormones. Physiological studies ofthe three hormones (RPCH, AKH I, and AKH II) and their syntheticanalogs show that they crossreact, i.e., they all exhibit pigment-concentratingactivity when tested on decapod crustaceans, adipokinetic activitywhen tested on locusts, and hyperglycemic activity when testedon cockroaches, although each of the hormones is more potentin its own system. Structure-function studies show, however,that quite different binding-site requirements exist for thehormones in activating their receptors on the various targettissues. The physiological specificity in their action thereforeseems to depend on a differential evolution of the hormone receptors.     

Calcium movements during pigment aggregation in freshwater shrimp chromatophores

Pigment granule migration within crustacean chromatophores provides an excellent model with which to investigate cytoplasmic movements, given the antagonistic, neurosecretory peptide regulation of granule translocation, and the absence of innervation in these large, brightly colored cells. Red pigment-concentrating hormone (RPCH) induces pigment aggregation in shrimp chromatophores via an increase in intracellular Ca2+; however, how this increase is brought about is not known. To examine the putative Ca2+ movements leading to pigment translocation in red, ovarian chromatophores of the freshwater shrimp, Macrobrachium olfersii, this study manipulates intra- and extracellular Ca2+ employing ER Ca2+-ATPase inhibitors, ryanodine-sensitive, ER Ca2+ channel blockers, and EDTA/EGTA-buffered A23187/Ca2+-containing salines. Our findings reveal that during pigment aggregation, cytosolic Ca2+ apparently increases from an intracellular source, the abundant SER, loaded by the SERCA and released through ryanodine-sensitive receptor/channels, triggered by capacitative calcium influx and/or calcium-induced calcium release mechanisms. Aggregation also depends on external calcium, which may modulate RPCH/receptor coupling. Such calcium-regulated pigment movements form the basis of a complex system of chromatic adaptation, which confers selective advantages like camouflage and protection against ultra-violet radiation to this palaemonid shrimp.