Isolation of xanthophores from the goldfish (Carassius auratus L.)

Summary A method is described for the isolation of milligram quantities of viable, hormone-responsive xanthophores from goldfish scales. The preparations are typically 70 to 90% pure and are useful for biochemical analyses or for establishing primary cultures. Preliminary results of this study were reported at the 11th International Pigment Cell Conference. This research was supported by Grant AM-13724 from the U.S. Public Health Service.     

Actin microfilaments in melanophores of Fundulus heteroclitus

Summary In melanophores of Fundulus heteroclitus, hormone-stimulated melanosome aggregation is accompanied by cytoplasmic flow from the cellular processes to the perikaryon, and reversal of these events takes place upon hormone-induced melanosome dispersion. These cells contain parallel arrays of microtubules, the majority of which are located in the perikaryon and in cortical regions of the processes. Studies with heavy meromyosin binding demonstrated two types of actin filaments: 1) a decorated meshwork of filaments similar to those usually found in close association with plasma membranes, and 2) filaments decorated in a manner similar to that of stress fibers. There is an apparent increase in the amount of filaments during melanosome aggregation. These results are discussed in relation to intracellular movement.Supported, in part, by grants AM-5384 and AM-13724 from U.S.P.H.S., and grant 234046 from the Japanese Ministry of Education     

Phosphorylation of the carotenoid droplet protein p57 by the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase and the effect of fluoride

We have previously shown that the dispersion and aggregation of carotenoid droplets in goldfish xanthophores are regulated, respectively, by phosphorylation and dephosphorylation of a carotenoid droplet protein p57. There is a basal level of p57 phosphorylation of p57 in unstimulated cells, which is greatly stimulated by adrenocorticotropic hormone (ACTH) or cyclic adenosine monophosphate (cAMP) acting via cAMP-dependent protein kinase. We have also observed that, in permeabilized xanthophores, pigment dispersion can be induced when cAMP is replaced by fluoride. Since p57 has multiple phosphorylation sites, there is the question of whether all p57 phosphorylation is by cAMP-dependent protein kinase or whether phosphorylation by cAMP-independent protein kinase coupled with inhibition of phosphatase activity by fluoride can replace cAMP-dependent protein kinase and that the ability of fluoride to replace cAMP for pigment dispersion in permeabilized cells is probably due to activation of adenylcyclase. We also show that ACTH causes an approximately threefold increase in the level of cAMP in these cells.     

cAMP-independent and cAMP-dependent protein phosphorylations by isolated goldfish xanthophore cytoskeletons: evidence for the association of cytoskeleton with a carotenoid droplet protein

Triton-insoluble cytoskeleton of nonpigment cells has bound protein kinase that phosphorylates, with or without added cAMP, tubulins and the intermediate filament proteins p60, p56, p53, and p45a to give multiple charge variants. In the absence of 8-Br-cAMP, Triton-insoluble cytoskeletons from xanthophores also phosphorylate p60, p56, and p45a, but not p53; tubulin phosphorylation may also be reduced. In the presence of 8-Br-cAMP, p53, as well as several other peptides, are phosphorylated. One of these latter peptides was identified as the carotenoid droplet (pigment organelle) protein p57, whose phosphorylation and dephosphorylation precede pigment dispersion and aggregation respectively (Lynch et al.: J. Biol. Chem. 261:4204-4211, 1986). The amount of pp57 produced depends on the state of pigment distribution in the xanthophores used to prepare the cytoskeletons for labeling. With cytoskeletons from xanthophores with aggregated pigment, pp57 is a major labeled phosphoprotein seen in two-dimensional gels. With cytoskeletons prepared from xanthophores with dispersed pigment, the yield of labeled pp57 is greatly reduced (by at least 90%). Together with earlier results, we propose that, in the aggregated state, p57 serves to bind carotenoid droplets to the cytoskeletons, most likely the microtubules. The significance of other cAMP-dependent phosphorylation reactions is unknown but may be related to cAMP-induced cytoskeleton rearrangement in intact xanthophores.     

Regulation of pigment organelle translocation. I. Phosphorylation of the organelle-associated protein p57

Treatment of goldfish xanthophores with adrenocorticotropin (ACTH) or cyclic AMP (cAMP) induces the centrifugal movement of their pigment organelles from the center of the cells. Using purified xanthophores pulse labeled with 32Pi, we have shown that the dispersion of the organelles is accompanied by the phosphorylation of a pair of polypeptides, termed p57. After fractionation on sucrose gradients, nearly all of the p57 is found associated with the pigment organelles. The phosphorylation induced by ACTH or cAMP apparently occurs at multiple sites on p57. The minimal effective doses of ACTH or cAMP required to induce full pigment dispersion also fully stimulate the phosphorylation of p57. Increased phosphorylation of p57 is detectable within a minute after stimulating the cells and appears to be near completion during the early phases of pigment dispersion. Upon withdrawal of ACTH, these events are reversed; the pigment organelles reaggregate toward the center of the cells and p57 is dephosphorylated. Again, dephosphorylation commences soon after ACTH is withdrawn and is complete before the organelles have completely reaggregated. These results suggest a novel mechanism for governing the movement of these organelles which acts on the organelles themselves through the phosphorylation and dephosphorylation of p57.     

Changes in the distribution of melanophores and xanthophores inTriturus alpestris embryos during their transition from the uniform to banded pattern

Summary The change in distribution of melanophores from stage 28+ (uniform melanophore pattern) to stage 34 (banded melanophore pattern) and the participation of xanthophores in these changes has been investigated inTriturus alpestris embryos by studying the social behaviour of single cells. While melanophores are clearly visible from outside the embryo at stage 28+, xanthophores cannot be recognized from the outside until after stage 34. In ultrathin sections of stage 34 embryos, xanthophores are observed alternating with melanophores in a zonal distribution (Epperlein 1982). Using detached pieces of dorsolateral trunk skin, which retain their chromatophores after detachment, the entire distribution of melanophores and xanthophores can be visualized in a scanning electron microscope (SEM). In ambiguous cases (early stages), cells were reprocessed for transmission electron microscopy (TEM) and the presence of the characteristic pigment organelles was assessed. In addition, xanthophores were specifically identified by pteridine fluorescence with dilute ammonia. Pteridines were also identified chromatographically in skin homogenates. The combination of these methods allowed precise identification and quantitative determination of melanophores and xanthophores. Both cell types were present as codistributed, biochemically differentiated cells at stage 28+. Changes in the pattern up to stage 34 were due to the rearrangement at the epidermal-mesodermal interface of a relatively fixed number of melanophores which became preferentially localised at the dorsal somite edge and at the lateral plate mesoderm, and to the distribution of an increasing number of xanthophores to subepidermal locations in the dorsal fin and between the melanophore bands. Concomitant was an increase in the thickness of the epidermal basement membrane and a change in shape of chromatophores from elongate via stellate to rosette shaped, which may be correlated with a shift from migratory to sessile phases.     

Pigment cell differentiation in the fire-bellied toad,Bombina orientalis. I. Structural,chemical, and physical aspects of the adult pigment pattern

Wild-collected adults of Bombina orientalis are bright green dorsally and red to red-orange ventrally. As a prelude to an analysis of the differentiation of pigment cells in developing B. orientalis, we describe structural and chemical aspects of the fully differentiated pigment pattern of the “normal” adult. Structurally, differences between dorsal green and ventral red skin are summarized as follows: (1) Dorsal green skin contains a “typical” dermal chromatophore unit comprised of melanophores, iridophores, and xanthophores. Red skin contains predominantly carotenoid-containing xanthophores (erythrophores), and skin from black spot areas contains only melanophores. (2) In ventral red skin, there is also a thin layer of deep-lying iridophores that presumably are not involved in the observed color pattern. (3) Xanthophores of red and green skin are morphologically distinguishable from each other. Dorsal skin xanthophores contain both pterinosomes and carotenoid vesicles; ventral skin xanthophores contain only carotenoid vesicles. Carotenoid vesicles in dorsal xanthophores are much larger but less electron dense than comparable structures in ventral xanthophores. The presence of carotenes in ventral skin accounts for the bright red-orange color of the belly of this frog. Similar pigments are also present in green skin, but in smaller quantities and in conjunction with both colored (yellow) and colorless pteridines. From spectral data obtained for xanthophore pigments and structural data obtained from the size and arrangement of reflecting platelets in the iridophore layer, we attempt to explain the phenomenon of observed green color in B. orientalis.     

Tetraspanin 3c requirement for pigment cell interactions and boundary formation in zebrafish adult pigment stripes

Skin pigment pattern formation in zebrafish requires pigment‐cell autonomous interactions between melanophores and xanthophores, yet the molecular bases for these interactions remain largely unknown. Here, we examined the dali mutant that exhibits stripes in which melanophores are intermingled abnormally with xanthophores. By in vitro cell culture, we found that melanophores of dali mutants have a defect in motility and that interactions between melanophores and xanthophores are defective as well. Positional cloning and rescue identified dali as tetraspanin 3c (tspan3c), encoding a transmembrane scaffolding protein expressed by melanophores and xanthophores. We further showed that dali mutant Tspan3c expressed in HeLa cell exhibits a defect in N‐glycosylation and is retained inappropriately in the endoplasmic reticulum. Our results are the first to identify roles for a tetraspanin superfamily protein in skin pigment pattern formation and suggest new mechanisms for the establishment and maintenance of zebrafish stripe boundaries.     

Purification of anterogin, a protein factor necessary for the dispersion of carotenoid droplets in permeabilized xanthophores of goldfish

We reported previously that the dispersion of carotenoid droplets in permeabilized xanthophores requires cAMP, ATP, and a cytosolic factor present in several secretory tissues as well as in xanthophores. We have now purified this factor from beef liver to apparent/near homogeneity. It appears to be a heterodimer with Mr approximately 125,000. The purified factor has little or no ATPase activity, with or without the presence of actin. Nor does it stimulate the ATPase activity of carotenoid droplets. Its exact function in carotenoid droplet dispersion is thus unclear. Since dispersion of carotenoid droplets is an anterograde translocation, we propose the name anterogin for this protein. We also report that yeast cytosol has anterogin activity.     

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.     

Dermal and epidermal chromatophores of the Antarctic teleost Trematomus bernacchii

The physiological response and ultrastructure of the pigment cells of Trematomus bernacchii, an Antarctic teleost that lives under the sea ice north of the Ross Ice Shelf, were studied. In the integument, two types of epidermal chromatophores, melanophores and xanthophores, were found; in the dermis, typically three types of chromatophores--melanophores, xanthophores, and iridophores--were observed. The occurrence of epidermal xanthophore is reported for the first time in fish. Dermal melanophores and xanthophores have well-developed arrays of cytoplasmic microtubules. They responded rapidly to epinephrine and teleost melanin-concentrating hormone (MCH) with pigment aggregation and to theophylline with pigment dispersion. Total darkness elicited pigment aggregation in the majority of dermal xanthophores of isolated scales, whereas melanophores remained dispersed under both light and dark conditions. Pigment organelles of epidermal and dermal xanthophores that translocate during the pigmentary responses are carotenoid droplets of relatively large size. Dermal iridophores containing large reflecting platelets appeared to be immobile.     

Actin-dependent carotenoid droplet dispersion in permeabilized cultured goldfish xanthophores

Organelle translocations are essential cellular processes. Although much progress has been made with regards to microtubule-dependent organelle translocations, little is known about actin-dependent organelle translocation(s) except cytoplasmic streaming in Nitella. On the other hand, there is indirect evidence that actin-dependent organelle translocation may be involved in secretion. We now present evidence that the dispersion of the pigment organelles carotenoid droplets in goldfish xanthophores is apparently actin dependent and that this process may be related to secretory processes. We show that, in digitonin-permeabilized goldfish xanthophores, the pigment organelles can be induced to disperse by a combination of cAMP, ATP, and xanthophore cytosol. This induced dispersion is inhibited by DNase I, phalloidin, or anti-actin, but not by anti-tubulin or anti-intermediate filament proteins, suggesting a dependence on F-actin. Since the dispersion of carotenoid droplets and secretion both involve outward translocation of organelles, we tested the possibility that cytosols of secretory tissues have similar activity. Such activity was indeed found in different tissues, apparently in parallel with the secretory activity of the tissues, suggesting that pigment dispersion in xanthophores and some secretory processes may share a common component.     

Medaka double mutants for color interfere and leucophore free: characterization of the xanthophore–somatolactin relationship using the leucophore free gene

Somatolactin (SL) plays an essential role in body-color regulation in medaka and is encoded by the color interfere (ci) locus. The ci mutant fish possess constitutively increased numbers of leucophores and a concomitant decrease in visible xanthophores. However, the mechanism of action of SL on these cell types, and the role of SL in body-color regulation in other species, is unknown. In this study, we verified an SL–xanthophore relationship in ci mutant fish using the leucophore free (lf) gene. Histological observation of lf larvae indicated that these mutants do not possess differentiated leucophores. The ci–lf double mutant, whose genotype was confirmed using DNA markers, lacked leucophores; however, the number of xanthophores remained low, demonstrating that leucophores are not necessary for mediating SL signaling to xanthophores. This finding suggests a conserved function for SL in xanthophore regulation across species, rather than the evolution of a medaka-specific and leucophore-dependent role of SL in body-color regulation. Our results also demonstrate that the lf gene has an indispensable role in leucophore development epistatic to SL signaling. The lf gene has not been cloned. The high-resolution recombination map surrounding the lf locus constructed in this study, together with medaka whole genome sequences that will be released soon, will allow the rapid cloning of the lf gene by forward genetic approaches.     

N-CAM and N-Cadherin Are Specifically Expressed in Xanthophores,but not in the Other Types of Pigment Cells,Melanophores, and Iridiphores

Little is known about cell-cell communication in pigment cells, whereas a number of signalling molecules have been implicated to control their migration, differentiation, and proliferation. We set out to investigate the expression of cell adhesion molecules (CAMs) in the three different types of pigment cells in poikilotherms, Oryzias latipes and Xenopus laevis. In the present experiments, the expression of N-CAM and N-cadherin in the pigment cells in vitro was examined by immunocytochemistry. Melanophores and xanthophores were isolated and cultured from scales or skins, while iridophores were harvested from skins or peritoneum. The results showed that N-CAM and N-cadherin were specifically expressed in xanthophores, but not in melanophores or iridophores in both O.latipes and X.laevis. N-CAM and N-cadherin basically colocalized in the restricted regions of xanthophores, although the N-cadherin-expressed region was broader than the N-CAM-expressed region in the same cell. The incidence of N-cadherin expression was higher than that of N-CAM expression. N-CAM and N-cadherin were expressed at the tip or the base of dendrites, or at the edge between dendrites in dendritic xanthophores. N-CAM and N-cadherin usually localized in small and narrow regions of xanthophores. This distribution pattern was essentially similar in xanthophores with round morphology, which exhibited spot, band, or semicircular immunoreactive regions on the peripheral edge of the cells. The difference in the distribution of pigment granules within the cells, culture period, fixatives, or immunofluorescent markers used in the experiments did not alter the immunostaining pattern.     

Regulation of the distribution of carotenoid droplets in goldfish xanthophores and possible implication to secretory processes

In goldfish xanthophores, the formation of pigment aggregate requires: 1) that a pigment organelle (carotenoid droplet) protein p57 be in the unphosphorylated state; 2) that self-association of pigment organelles occur in a microtubule-independent manner; and 3) that pigment organelles via p57 associate with microtubules. In the fully aggregated state, the pigment organelles are completely stationary. Pigment dispersion is initiated by activation of a cAMP-dependent protein kinase, which phosphorylates p57 and allows pigment dispersion via an active process dependent on F-actin and a cytosolic factor. This factor is not an ATPase, and its function is unknown. However, its abundance in different tissues parallels secretory activity of the tissues, suggesting a similarity between secretion and pigment dispersion in xanthophores. The identity of the motor for pigment dispersion is unclear. Experimental results show that pigment organelles isolated from cells with dispersed pigment have associated actin and ATPase activity comparable to myosin ATPase. This ATPase is probably an organelle protein of relative molecular mass approximately 72,000, and unlikely to be an ion pump. Isolated pigment organelles without associated actin have 5x lower ATPase activity. Whether this organelle ATPase is the motor for pigment dispersion is under investigation. The process of pigment aggregation is poorly understood, with conflicting results for and against the involvement of intermediate filaments.     

The Pteridine Pathway in Zebrafish: Regulation and Specification during the Determination of Neural Crest Cell‐Fate

This review describes pteridine biosynthesis and its relation to the differentiation of neural crest derivatives in zebrafish. During the embryonic development of these fish, neural crest precursor cells segregate into neural elements, ectomesenchymal cells and pigment cells; the latter then diversifying into melanophores, iridophores and xanthophores. The differentiation of neural cells, melanophores, and xanthophores is coupled closely with the onset of pteridine synthesis which starts from GTP and is regulated through the control of GTP cyclohydrolase I activity. De novo pteridine synthesis in embryos of this species increases during the first 72‐h postfertilization, producing H₄biopterin, which serves as a cofactor for neurotransmitter synthesis in neural cells and for tyrosine production in melanophores. Thereafter, sepiapterin (6‐lactoyl‐7,8‐dihydropterin) accumulates as yellow pigment in xanthophores, together with 7‐oxobiopterin, isoxanthopterin and 2,4,7‐trioxopteridine. Sepiapterin is the key intermediate in the formation of 7‐oxopteridines, which depends on the availability of enzymes belonging to the xanthine oxidoreductase family. Expression of the GTP cyclohydrolase I gene (gch) is found in neural cells, in melanoblasts and in early xanthophores (xanthoblasts) of early zebrafish embryos but steeply declines in xanthophores by 42‐h postfertilization. The mechanism(s) whereby sepiapterin branches off from the GTP‐H₄biopterin pathway is currently unknown and will require further study. The surge of interest in zebrafish as a model for vertebrate development and its amenability to genetic manipulation provide powerful tools for analysing the functional commitment of neural crest‐derived cells and the regulation of pteridine synthesis in mammals.     

Different distribution of melanophores and xanthophores in early tailbud and larval stages inTriturus alpestris

Summary The subepidermal distribution of xanthophores and melanophores is investigated in embryos ofTriturus alpestris with a uniform (stage 28+) and a banded melanophore pattern (stage 35/36). In ultrathin head and trunk sections from stage 35/36 embryos which externally show longitudinal dorsal and lateral melanophore bands in the trunk and less compact continuations of the dorsal bands in the head, xanthophores were discovered in addition to melanophores. Melanophores contain melanosomes while xanthophores which are not externally visible, are recognized by their pterinosomes. Both chromatophore cell types are mutually exclusively distributed on the epidermal basement membrane (bm). Mesenchymal cells seemed not to be able to replace them, except on the bm of the corneal epithelium where there were only mesenchymal cells. In head and trunk sections from stage 28+ embryos which externally show a distribution of uniformly scattered melanophores on the dorsolateral halves, melanophores were found on the dorsolateral neural crest migration route. No epidermal bm was present and xanthophores were undetectable. In ventrolateral and ventral portions of embryos of both stages no chromatophores occurred. This investigation defines the histological localization of melanophores and xanthophores in embryos with a typical uniform and banded melanophore arrangement; a subsequent study analyzes when xanthophores appear and how they arrange with melanophores in alternating zones.     

Chromatophores and color change in the lizard,Anolis carolinensis

Summary The skin of the lizard, Anolis carolinensis, changes rapidly from bright green to a dark brown color in response to melanophore stimulating hormone (MSH). Chromatophores responsible for color changes of the skin are xanthophores which lie just beneath the basal lamina containing pterinosomes and carotenoid vesicles. Iridophores lying immediately below the xanthophores contain regularly arranged rows of reflecting platelets. Melanophores containing melanosomes are present immediately below the iridophores. The ultrastructural features of these chromatophores and their pigmentary organelles are described. The color of Anolis skin is determined by the position of the melanosomes within the melanophores which is regulated by MSH and other hormones such as norepinephrine. Skins are green when melanosomes are located in a perinuclear position within melanophores. In response to MSH, they migrate into the terminal processes of the melanophores which overlie the xanthophores above, thus effectively preventing light penetration to the iridophores below, resulting in skins becoming brown. The structural and functional characteristics of Anolis chromatophores are compared to the dermal chromatophore unit of the frog.This study was supported in part by GB-8347 from the National Science Foundation.Contribution No. 244, Department of Biology, Wayne State University.The authors are indebted to Dr. Joseph T. Bagnara for his encouragement during the study and to Dr. Wayne Ferris for his advice and the use of his electron microscope laboratory.     

Unusual Leucophore‐Like Cells Specifically Appear in the Lineage of Melanophores in the Periodic Albino Mutant of Xenopus laevis

In the periodic albino mutant (a^p/a^p) of Xenopus laevis, peculiar leucophore‐like cells appear in the skins of tadpoles and froglets, whereas no such cells are observed in the wild‐type (+/+). These leucophore‐like cells are unusual in (1) appearing white, but not iridescent, under incident light, (2) emitting green fluorescence under blue light, (3) exhibiting pigment dispersion in the presence of α‐melanocyte stimulating hormone (αMSH), and (4) containing an abundance of bizarre‐shaped, reflecting platelet‐like organelles. In this study, the developmental and ultrastructural characteristics of these leucophore‐like cells were compared with melanophores, iridophores and xanthophores, utilizing fluorescence stereomicroscopy, and light and electron microscopy. Staining with methylene blue, exposure to αMSH, and culture of neural crest cells were also performed to clarify the pigment cell type. The results obtained clearly indicate that: (1) the leucophore‐like cells in the mutant are different from melanophores, iridophores and xanthophores, (2) the leucophore‐like cells are essentially similar to melanophores of the wild‐type with respect to their localization in the skin and manner of response to αMSH, (3) the leucophore‐like cells contain many premelanosomes that are observed in developing melanophores, and (4) mosaic pigment cells containing both melanosomes specific to mutant melanophores and peculiar reflecting platelet‐like organelles are observed in the mutant tadpoles. These findings strongly suggest that the leucophore‐like cells in the periodic albino mutant are derived from the melanophore lineage, which provides some insight into the origin of brightly colored pigment cells in lower vertebrates.     

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.