Sdf1a patterns zebrafish melanophores and links the somite and melanophore pattern defects in choker mutants

Pigment pattern formation in zebrafish presents a tractable model system for studying the morphogenesis of neural crest derivatives. Embryos mutant for choker manifest a unique pigment pattern phenotype that combines a loss of lateral stripe melanophores with an ectopic melanophore ;collar' at the head-trunk border. We find that defects in neural crest migration are largely restricted to the lateral migration pathway, affecting both xanthophores (lost) and melanophores (gained) in choker mutants. Double mutant and timelapse analyses demonstrate that these defects are likely to be driven independently, the collar being formed by invasion of melanophores from the dorsal and ventral stripes. Using tissue transplantation, we show that melanophore patterning depends upon the underlying somitic cells, the myotomal derivatives of which--both slow--and fast-twitch muscle fibres--are themselves significantly disorganised in the region of the ectopic collar. In addition, we uncover an aberrant pattern of expression of the gene encoding the chemokine Sdf1a in choker mutant homozygotes that correlates with each aspect of the melanophore pattern defect. Using morpholino knock-down and ectopic expression experiments, we provide evidence to suggest that Sdf1a drives melanophore invasion in the choker mutant collar and normally plays an essential role in patterning the lateral stripe. We thus identify Sdf1 as a key molecule in pigment pattern formation, adding to the growing inventory of its roles in embryonic development.     

Homology and evolutionary novelty in the deployment of extracellular matrix molecules during pigment pattern formation in the salamanders Taricha torosa and T. rivularis (Salamandridae)

Salamander larvae exhibit a diverse array of pigment patterns shortly after hatching. Previous studies have identified roles for the extracellular matrix and lateral line sensory system in promoting the development of a phylogenetically common pattern of horizontal melanophore stripes. In contrast, salamanders in the genus Taricha exhibit evolutionarily derived pigment patterns and pattern-forming mechanisms. Taricha torosa larvae exhibit compact melanophore stripes that develop via redundant, lateral line-independent mechanisms, whereas T. rivularis larvae lack stripes and instead have melanophores uniformly distributed over the flank. In this study, I test roles for candidate patterning molecules of the extracellular matrix in promoting the development of species-specific pigment patterns in Taricha. I show that tenascin deposition is negatively correlated with melanophore distributions both intraspecifically and interspecifically: this matrix molecule is present where melanophores do not localize in T. torosa and is absent from these same regions where melanophores are abundant in T. rivularis. Embryological manipulations further indicate that transient expression of tenascin in a prospective interstripe region of T. torosa reflects a phylogenetically conserved effect of lateral line development. Finally, anti-laminin immunoreactivity is negatively correlated with melanophore distributions in T. torosa, and this species exhibits a general retardation of extracellular matrix development that may allow persistent, evolutionarily novel melanophore motility in this species. Together these findings identify tenascin and laminin, or molecules co-regulated with these matrix components, as candidates for promoting early larval pigment pattern development in Taricha.     

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.     

Biosensing of opioids using frog melanophores

Spectacular color changes of fishes, frogs and other lower vertebrates are due to the motile activities of specialized pigment containing cells. Pigment cells are interesting for biosensing purposes since they provide an easily monitored physiological phenomenon. Melanophores, containing dark brown melanin pigment granules, constitute an important class of chromatophores. Their melanin-filled pigment granules may be stimulated to undergo rapid dispersion throughout the melanophores (cells appear dark), or aggregation to the center of the melanophores (cells appear light). This simple physiological response can easily be measured in a photometer. Selected G protein coupled receptors can be functionally expressed in cultured frog melanophores. Here, we demonstrate the use of recombinant frog melanophores as a biosensor for the detection of opioids. Melanophores were transfected with the human opioid receptor 3 and used for opiate detection. The response to the opioid receptor agonist morphine and a synthetic opioid peptide was analyzed by absorbance readings in an aggregation assay. It was shown that both agonists caused aggregation of pigment granules in the melanophores, and the cells appeared lighter. The pharmacology of the expressed receptors was very similar to its mammalian counterpart, as evidenced by competitive inhibition by increasing concentrations of the opioid receptor inhibitor naloxone. Transfection of melanophores with selected receptors enables the creation of numerous melanophore biosensors, which respond selectively to certain substances. The melanophore biosensor has potential use for measurement of substances in body fluids such as saliva, blood plasma and urine.     

Studies on cellular adhesion of Xenopus laevis melanophores: pigment pattern formation and alteration in vivo by endogenous galactoside-binding lectin or its sugar hapten inhibitor

We have studied the development of Xenopus laevis tail melanophores and the effects on these cells on confrontation with endogenous X. laevis galactoside-binding lectin or its sugar hapten inhibitor thiodigalactoside (TDG). An initial population of unpigmented cells differentiates into melanophores on the dorsal surface of the neural tube, and on the dorsal and ventral apices of the myotomes, forming the larval pattern. Melanophores secondarily populate the flank, forming a spaced arrangement which is later transformed into a dorsal and ventral strip. A technique has been developed for confrontation of premigratory neural crest with purified lectin or TDG. These molecules impact on tail melanophores. With lectin treatment melanophore numbers decrease, and cell morphologies and arrangements change. TDG treatment, however, primarily affects pigment cell morphology. These results suggest that both galactoside-bearing receptors for this lectin and the lectin itself can affect melanophores in this species of frog.     

Ultrastructural changes in dermal melanophores in the process of pigment granule migration

The results of electron microscope investigations on dermal melanophores of Rana temporaria L. during migration of pigment granules are presented. It was shown that in comparison to the previous observations dermal melanophores are flat cells without branches. Ultrastructural differences have been demonstrated in dermal melanophores during migration of pigment granules. During melanosome dispersion membrane vesicle bodies are seen in the cytoplasm to be inserted in the melanophore membrane.     

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.     

Microtubules and pigment migration in the melanophores ofFundulus heteroclitus L.

Summary The dermal melanophores ofFundulus heteroclitus L. have been investigated by light and electron microscopy with the purpose of revealing the mechanisms controlling pigment migration. As predicted by earlier studies, the nerve endings of a double innervation were found adjacent to and in synaptic relation to the melanophore surface. Not expected were the large number of small pits or invaginations present in the cell surface. These appear identical to the so-called micropinocytotic vesicles found generally in cells of the vascular endothalium and smooth muscle. In chromatophores they are more reasonably interpreted as receptor sites for neurohormones than as uptake and transport mechanisms.Observations made on the kinetics of pigment migration within the processes of these melanophores indicate that the granules move along relatively fixed channels arranged parallel to the long axes of the processes. Examined at fine structure levels, the zones of cytoplasm around these channels are found to be populated by microtubules about 225 Å in diameter aligned parallel to the direction of pigment movement. These long slender elements are present in the processes regardless of whether the melanin is concentrated in the cell center or dispersed. It is reasoned from these and other observations that the microtubules function as cytoskeletal elements which help maintain the extended form of the melanophore arms and at the same time define the channels in which the pigment moves. The possible role of the tubule in generating the motive force for pigment migration is discussed.Supported by US Public Health Service Training Grant, 5 TIGM-707.     

Temporal and cellular requirements for Fms signaling during zebrafish adult pigment pattern development

Ectothermic vertebrates exhibit a diverse array of adult pigment patterns. A common element of these patterns is alternating dark and light stripes each comprising different classes of neural crest-derived pigment cells. In the zebrafish, Danio rerio, alternating horizontal stripes of black melanophores and yellow xanthophores are a prominent feature of the adult pigment pattern. In fms mutant zebrafish, however, xanthophores fail to develop and melanophore stripes are severely disrupted. fms encodes a type III receptor tyrosine kinase expressed by xanthophores and their precursors and is the closest known homologue of kit, which has long been studied for roles in pigment pattern development in amniotes. In this study we assess the cellular and temporal requirements for Fms activity in promoting adult pigment pattern development. By transplanting cells between fms mutants and either wild-type or nacre mutant zebrafish, we show that fms acts autonomously to the xanthophore lineage in promoting the striped arrangement of adult melanophores. To identify critical periods for fms activity, we isolated temperature sensitive alleles of fms and performed reciprocal temperature shift experiments at a range of stages from embryo to adult. These analyses demonstrate that Fms is essential for maintaining cells of the xanthophore lineage as well as maintaining the organization of melanophore stripes throughout development. Finally, we show that restoring Fms activity even at late larval stages allows essentially complete recovery of xanthophores and the development of a normal melanophore stripe pattern. Our findings suggest that fms is not required for establishing a population of precursor cells during embryogenesis but is required for recruiting pigment cell precursors to xanthophore fates, with concomitant effects on melanophore organization.     

Ultrastructure of the integumental melanophores of the coelacanth,Latimeria chalumnae

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

Studies of pigment transfer between Xenopus laevis melanophores and fibroblasts in vitro and in vivo1

Frog melanophores rapidly change colour by dispersion or aggregation of melanosomes. A long‐term colour change exists where melanosomes are released from melanophores and transferred to surrounding skin cells. No in vitro model for pigment transfer exists for lower vertebrates. Frog melanophores of different morphology exist both in epidermis where keratinocytes are present and in dermis where fibroblasts dominate. We have examined whether release and transfer of melanosomes can be studied in a melanophore‐fibroblast co‐culture, as no frog keratinocyte cell line exists. Xenopus laevis melanophores are normally cultured in conditioned medium from fibroblasts and fibroblast‐derived factors may be important for melanophore morphology. Melanin was exocytosed as membrane‐enclosed melanosomes in a process that was upregulated by α‐melanocyte‐stimulating hormone (α‐MSH), and melanosomes where taken up by fibroblasts. Melanosome membrane‐proteins seemed to be of importance, as the cluster‐like uptake pattern of pigment granules was distinct from that of latex beads. In vivo results confirmed the ability of dermal fibroblasts to engulf melanosomes. Our results show that cultured frog melanophores can not only be used for studies of rapid colour change, but also as a model system for long‐term colour changes and for studies of factors that affect pigmentation.     

Evolutionary diversification of pigment pattern in Danio fishes: differential fms dependence and stripe loss in D. albolineatus

The developmental bases for species differences in adult phenotypes remain largely unknown. An emerging system for studying such variation is the adult pigment pattern expressed by Danio fishes. These patterns result from several classes of pigment cells including black melanophores and yellow xanthophores, which differentiate during metamorphosis from latent stem cells of presumptive neural crest origin. In the zebrafish D. rerio, alternating light and dark horizontal stripes develop, in part, owing to interactions between melanophores and cells of the xanthophore lineage that depend on the fms receptor tyrosine kinase; zebrafish fms mutants lack xanthophores and have disrupted melanophore stripes. By contrast, the closely related species D. albolineatus exhibits a uniform pattern of melanophores, and previous interspecific complementation tests identified fms as a potential contributor to this difference between species. Here, we survey additional species and demonstrate marked variation in the fms-dependence of hybrid pigment patterns, suggesting interspecific variation in the fms pathway or fms requirements during pigment pattern formation. We next examine the cellular bases for the evolutionary loss of stripes in D. albolineatus and test the simplest model to explain this transformation, a loss of fms activity in D. albolineatus relative to D. rerio. Within D. albolineatus, we demonstrate increased rates of melanophore death and decreased melanophore migration, different from wild-type D. rerio but similar to fms mutant D. rerio. Yet, we also find persistent fms expression in D. albolineatus and enhanced xanthophore development compared with wild-type D. rerio, and in stark contrast to fms mutant D. rerio. These findings exclude the simplest model in which stripe loss in D. albolineatus results from a loss of fms-dependent xanthophores and their interactions with melanophores. Rather, our results suggest an alternative model in which evolutionary changes in pigment cell interactions themselves have contributed to stripe loss, and we test this model by manipulating melanophore numbers in interspecific hybrids. Together, these data suggest evolutionary changes in the fms pathway or fms requirements, and identify changes in cellular interactions as a likely mechanism of evolutionary change in Danio pigment patterns.     

Studies of pigment transfer between Xenopus laevis melanophores and fibroblasts in vitro and in vivo

Frog melanophores rapidly change colour by dispersion or aggregation of melanosomes. A long-term colour change exists where melanosomes are released from melanophores and transferred to surrounding skin cells. No in vitro model for pigment transfer exists for lower vertebrates. Frog melanophores of different morphology exist both in epidermis where keratinocytes are present and in dermis where fibroblasts dominate. We have examined whether release and transfer of melanosomes can be studied in a melanophore-fibroblast co-culture, as no frog keratinocyte cell line exists. Xenopus laevis melanophores are normally cultured in conditioned medium from fibroblasts and fibroblast-derived factors may be important for melanophore morphology. Melanin was exocytosed as membrane-enclosed melanosomes in a process that was upregulated by alpha-melanocyte-stimulating hormone (alpha-MSH), and melanosomes where taken up by fibroblasts. Melanosome membrane-proteins seemed to be of importance, as the cluster-like uptake pattern of pigment granules was distinct from that of latex beads. In vivo results confirmed the ability of dermal fibroblasts to engulf melanosomes. Our results show that cultured frog melanophores can not only be used for studies of rapid colour change, but also as a model system for long-term colour changes and for studies of factors that affect pigmentation.     

Frog melanophores cultured on fluorescent microbeads: biomimic-based biosensing

Melanophores are pigmented cells in lower vertebrates capable of quick color changes and thereby suitable as whole cell biosensors. In the frog dermis skin layer, the large and dark pigmented melanophore surrounds a core of other pigmented cells. Upon hormonal stimulation the black-brown pigment organelles will redistribute within the melanophore, and thereby cover or uncover the core, making complex color changes possible in the dermis. Previously, melanophores have only been cultured on flat surfaces. Here we mimic the three dimensional biological geometry in the frog dermis by culturing melanophores on fluorescent plastic microbeads. To demonstrate biosensing we use the hormones melatonin and alpha-melanocyte stimulating hormone (alpha-MSH) as lightening or darkening stimuli, respectively. Cellular responses were successfully demonstrated on single cell level by fluorescence microscopy, and in cell suspension by a fluorescence microplate reader and a previously demonstrated computer screen photo-assisted technique. The demonstrated principle is the first step towards "single well/multiple read-out" biosensor arrays based on suspensions of different selective-responding melanophores, each cultured on microbeads with distinctive spectral characteristics. By applying small amount of a clinical sample, or a candidate substance in early drug screening, to a single well containing combinations of melanophores on beads, multiple parameter read-outs will be possible.     

鳜皮肤图案中色素细胞的分布与排列

色素细胞是皮肤图案形成的基础,为了解鳜(Siniperca chuatsi)皮肤图案区域色素细胞的种类、分布及排列特征,采用光学显微镜与电子显微镜对鳜皮肤中图案区域、非图案区域及交界处皮肤的色素细胞进行显微及超显微结构观察。结果显示,鳜皮肤中含有黑色素细胞、黄色素细胞、红色素细胞及虹彩细胞,主要分布于表皮层和色素层。头部过眼条纹、躯干纵带、躯干斑块等图案区域皮肤表皮层与色素层均含有黑色素细胞,非图案区域仅表皮层含有少量黑色素细胞。躯干图案区域(纵带、斑块)皮肤色素层色素细胞分布层次明显,由外到内依次为黄色素细胞、红色素细胞、黑色素细胞和虹彩细胞,其中,虹彩细胞内反射小板较长,整齐水平排列;躯干非图案区域皮肤色素层由外到内依次为黄色素细胞、红色素细胞和虹彩细胞,其中,虹彩细胞内反射小板较短,无规则排列。头部过眼条纹色素层含有4种色素细胞,色素细胞数量较少,且无规则排列,其中,黑色素细胞内黑色素颗粒较大。交界处皮肤色素层黑色素细胞数量向非图案区域一侧逐渐减少,虹彩细胞数量逐渐增加。结果表明,鳜图案区域与非图案区域、不同图案区域的色素细胞分布与排列各不相同,本研究结果为鳜色素细胞图案化形成机...     

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.     

beta-Adrenergic receptor subtypes in melanophores of the marine gobies Tridentiger trigonocephalus and Chasmichthys gulosus.

The subtype of beta-adrenergic receptors in melanophores of the marine gobies Tridentiger trigonocephalus and Chasmichthys gulosus was studied. Pigment of denervated melanophores in isolated, split caudal fins was preliminarily aggregated by incubating the specimens in a physiological saline containing 10 microM phentolamine and 30-100 microM verapamil or 2-10 nM melatonin, and the responses of the melanophores to a beta-adrenergic agonist added to the incubating medium were recorded photoelectrically. The beta-adrenergic agonists noradrenaline, adrenaline, isoproterenol, salbutamol and, dobutamine were all effective in evoking a dispersion of melanophore pigment in the presence of phentolamine and verapamil or melatonin. The pigment-dispersing effect of noradrenaline (beta 1-selective agonist) was inhibited by metoprolol (beta 1-selective antagonist), propranolol,- and butoxamine. Whereas, the effect of salbutamol (beta 2-selective agonist) was hardly inhibited by metoprolol, though it was considerably inhibited by propranolol and ICI-118551. It was estimated that beta 1- and beta 2-adrenergic receptors coexist at ratios of 8.6:91.4, in the melanophore of Tridentiger trigonocephalus, and 25:75, in the melanophore of Chasmichthys gulosus, through the analyses of Hofstee plots of the effects of the beta-adrenergic drugs. It was suggested that the relation between the pigment-dispersing effect of a beta-adrenergic agonist on the melanophores and the concentration of the drug follows mass action kinetics, when the effect is mainly caused by the activation of beta 2-adrenergic receptors of the melanophores. However, when it is mainly caused by the activation of beta 1-adrenergic receptors of the melanophores, the relation does not follow mass action kinetics.     

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 alpha-melanocyte stimulating hormone (alphaMSH), 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 alphaMSH, 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 alphaMSH, (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.     

Long-term cultivation of amphibian melanophores. In vitro ageing and spontaneous transformation to a continuous cell line

Pure melanophore populations isolated from the tail skin of the tadpole, Rana catesbeiana, were mass cultured for a period of 2-3 years. All cell lines of amphibian melanophores studied exhibited growth crisis (in vitro ageing) followed by spontaneous transformation to a continuous cell line, as shown by changes in growth characteristics in mass culture and in clone culture, by the appearance of the cells, and by measurements of cell volumes. Even after becoming a continuous cell line, amphibian melanophores continued to have a diploid chromosome number (2n = 26) in three of four cell lines examined. The chromosome mode in one cell line, however, changed to thirty. Measurement of melanin dispersion after the addition of alpha-melanocyte-stimulating hormone suggested that the mechanism for melanin dispersion in melanophores changed during in vitro ageing.     

The origin and development of retinal pigment cells and melanophores analyzed by Xenopus black-white chimeras

At the 16 cell stage, three kinds of borealis–laevis and eight kinds of laevis–laevis chimeric embryos were produced by replacing a particular blastomere of albino embryos of Xenopus laevis with that of wild-type embryos of X. borealis or X. laevis , and then leaving the embryos to develop into frogs.
In the borealis–laevis chimera frogs, we found that all the melanized cells (retinal pigment cells and melanophores) were derived from a transplanted wild-type blastomere with a nuclear marker of X. borealis and that all the albino-mutant cells derived from the host did not become melanized. Thus, retinal pigment cells and melanophores differentiated according to their own genotype. We then examined the origin of these two types of cells, using melanin as a cell-marker in the borealis–laevis and laevis–laevis chimeras.
Retinal pigment cells derive from A1 (dorso-animal) and A2 (latero-animal) blastomeres. A1 of one side contributes to retinal pigment cells in both eyes. Though the blastomeres of one side contribute to the formation of bilateral melanophores, the major contribution is to melanophores of the same side. A1, A2 and V2 (latero-vegetal) form the anterior part of the neural fold, and A2 and V2 contribute to melanophores of the head region. The most anterior part of the neural fold derived from A1 does not make a significant contribution to melanophores. Though V2 is a vegetal blastomere, it forms the anterior part of the neural fold by upward movement against the downward movement for gastrulation. A3 forms the middle and posterior parts of the neural fold and contributes to melanophores of the trunk and hindlimbs. Melanophores of hindlimbs also come from A2, A4 and V2. It is to be noted that A4 contributes to melanophores of hindlimbs, despite no apparent contribution to the neural fold.
Development of the retinal pigment cells and melanophores is discussed from the point of pigmentation patterns of the chimeras.