Stimulation of Cultured Iridophores by Amphibian Ventral Conditioned Medium

That the ventral integument of adult frogs (Rana pipiens) contains factor(s) that stimulate iridophore expression (adhesion, morphologic appearance, proliferation) was demonstrated on iridophores derived from tadpoles of R. pipiens and Pachymedusa dacnicolor, and maintained in primary culture in a growth medium based upon Leibovitz's L-15. Experimental growth medium (VCM) conditioned by a one-hour exposure to pieces of ventral skin of adult R. pipiens induced iridophores to assume a broad and stellate appearance, to form confluent sheets, and to proliferate over a nine-day period. Iridophores in control medium assumed long thin profiles, detached easily, and exhibited no signs of proliferation. Unknown cells containing reflecting platelets and unusual other organelles appeared uniquely in chromatophore cultures of P. dacnicolor in VCM. The intense stimulation of iridophore expression in VCM is consistent with the known inhibitory effect of this medium on melanization and with its purported role in the determination of dorsal/ventral pigment patterns of amphibians. The results are discussed in terms of a prevailing theory about pigment cell origins and development.     

The dermal chromatophore unit

Rapid color changes of amphibians are mediated by three types of dermal chromatophores, xanthophores, iridophores, and melanophores, which comprise a morphologically and physiologically distinct structure, the dermal chromatophore unit. Xanthophores, the outermost element, are located immediately below the basal lamella. Iridophores, containing light-reflecting organelles, are found just beneath the xanthophores. Under each iridophore is found a melanophore from which processes extend upward around the iridophore. Finger-like structures project from these processes and occupy fixed spaces between the xanthophores and iridophores. When a frog darkens, melanosomes move upward from the body of the melanophore to fill the fingers which then obscure the overlying iridophore. Rapid blanching is accomplished by the evacuation of melanosomes from these fingers. Pale coloration ranging from tan to green is provided by the overlying xanthophores and iridophores. Details of chromatophore structure are presented, and the nature of the intimate contact between the chromatophore types is discussed.     

Intrinsic Pigment-Cell Stimulating Activity in the Catfish Integument

In keeping with the concept that local factors in the vertebrate integument affect the expression of pigment cells, the present study was directed toward demonstrating the existence of such factors in the skin of the channel catfish, Ictalurus punctatus. This species has a dark dorsal surface in marked contrast to an almost white midventral surface. Pieces of skin from these two surfaces were used to condition culture media, which were in turn bioassayed using the Xenopus neural tube explant system (Fukuzawa and Ide, 1988, Dev. Biol. 129:25). A certain number of neural crest cells grow out from the explant, and many of these are melanized in a culture medium of Steinberg's basic salt solution (BSS). When the BSS was conditioned with either dorsal or ventral skin, a profound increase in both the number of crest cells emigrated from the neural tubes and the percentage of melanized cells was observed. The effects of dorsal skin were stronger than those of ventral skin and were evident on a dose/response basis. Initial fractionation of conditioned BSS with DEAE ion exchange chromatography produced fractions of particular potency in the stimulation of melanogenesis. A similarly conditioned medium based upon Leibovitz's L-15 was used in the primary culture of mature chromatophores, namely, melanophores, iridophores, and xanthophores from tadpoles of Rana pipiens. Both dorsal and ventral conditioned media stimulated iridophores and xanthophores, but seemed to have little or no effect on tadpole melanophores. A melanization inhibiting factor (MIF) from the ventral surface of adult frogs has been suggested as the basis for the light colored ventrum of amphibians, and although the present experiments were not designed to study catfish MIF, the possible existence of such a factor in this species was supported by the results. The total results of this investigation are discussed in the light of the possible presence of a melanization inhibiting factor (MIF) of greater prevalence in the ventrum and a melanization stimulatory factor (MSF) of greater prevalence in the dorsal integument. It is suggested that the light-colored ventral surface of the catfish and other poikilotherms may result from the presence of higher levels of MIF than MSF. Thus, the expression of melanophores is inhibited while that of iridophores is enhanced. In contrast, higher levels of MSF over MIF in the dark dorsal surface would result in melanophore stimulation and inhibition of iridophore expression.     

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

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

Zebrafish puma mutant decouples pigment pattern and somatic metamorphosis

The genetic and developmental bases for trait expression and variation in adults are largely unknown. One system in which genes and cell behaviors underlying adult traits can be elucidated is the larval-to-adult transformation of zebrafish, Danio rerio. Metamorphosis in this and many other teleost fishes resembles amphibian metamorphosis, as a variety of larval traits (e.g., fins, skin, digestive tract, sensory systems) are remodeled in a coordinated manner to generate the adult form. Among these traits is the pigment pattern, which comprises several neural crest-derived pigment cell classes, including black melanophores, yellow xanthophores, and iridescent iridophores. D. rerio embryos and early larvae exhibit a relatively simple pattern of melanophore stripes, but this pattern is transformed during metamorphosis into the more complex pattern of the adult, consisting of alternating dark (melanophore, iridophore) and light (xanthophore, iridophore) horizontal stripes. While it is clear that some pigment cells differentiate de novo during pigment pattern metamorphosis, the extent to which larval and adult pigment patterns are developmentally independent has not been known. In this study, we show that a subset of embryonic/early larval melanophores persists into adult stages in wild-type fish; thus, larval and adult pigment patterns are not completely independent in this species. We also analyze puma mutant zebrafish, derived from a forward genetic screen to isolate mutations affecting postembryonic development. In puma mutants, a wild-type embryonic/early larval pigment pattern forms, but supernumerary early larval melanophores persist in ectopic locations through juvenile and adult stages. We then show that, although puma mutants undergo a somatic metamorphosis at the same time as wild-type fish, metamorphic melanophores that normally appear during these stages are absent. The puma mutation thus decouples metamorphosis of the pigment pattern from the metamorphosis of many other traits. Nevertheless, puma mutants ultimately recover large numbers of melanophores and exhibit extensive pattern regulation during juvenile development, when the wild-type pigment pattern already would be completed. Finally, we demonstrate that the puma mutant is both temperature-sensitive and growth-sensitive: extremely severe pigment pattern defects result at a high temperature, a high growth rate, or both; whereas a wild-type pigment pattern can be rescued at a low temperature and a low growth rate. Taken together, these results provide new insights into zebrafish pigment pattern metamorphosis and the capacity for pattern regulation when normal patterning mechanisms go awry.     

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.     

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.     

Extracellular matrix components influence the survival of adult cardiac myocytes in vitro

Calcium-tolerant myocytes were isolated from adult rat hearts by collagenase perfusion and plated on various substrates in serum-free medium and their adhesion to various extracellular matrix (ECM) components was determined. The myocytes attached readily to dishes coated with collagen type IV (C-IV), laminin (LN), and to fetal bovine serum (FBS) in a manner dependent on the concentration of the components. Substantially fewer myocytes adhered to dishes coated with fibronectin (FN) or to uncoated plastic dishes. Cells adhered equally well to dishes coated with C-IV, LN and FBS within 1-4 h. However, when examined after 2 weeks in culture it was found that only C-IV and LN could support survival of the attached myocytes, and when cultured on C-IV or LN the myocytes were spread and had formed a dense monolayer. The actin filaments had at this time reorganized linearly along the long axis of the cell and the myocytes contracted spontaneously. Rabbit antibodies were raised against myocyte membranes and their ability to inhibit attachment to ECM components was studied. Purified IgG inhibited attachment to C-IV, while having only a minor effect on attachment to LN. These data are compatible with the presence of a specific cell surface component(s) that interacts with ECM substrates and influences cell shape and possibly thereby influences cellular functions.     

Differentiation of neural crest cells of Xenopus laevis in clonal culture

Clonal cultures were performed with the use of neural crest cells and their derivatives, chromatophores, from Xenopus laevis in order to elucidate the state of commitment in early embryogenesis. Neural crest cells that outgrew from neural tube explants were isolated and plated at clonal density. Cloned neural crest cells differentiated and gave rise to colonies that consisted of 1) only melanophores, 2) only xanthophores, or 3) melanophores and xanthophores. Xanthophores and iridophores, which differentiated in vitro, were also isolated and cloned. Cloned xanthophores proliferated in a stable fashion and did not lose their properties. On the other hand, cloned iridophores converted into melanophores as they proliferated. These results suggest that there is heterogeneity in the state of commitment of neural crest cells immediately after migration with regard to chromatophore differentiation and that iridophore determination is relatively labile (at least in vitro), whereas melanophore and xanthophore phenotypes are stable.     

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.     

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.     

Modulation by extracellular matrices of monooxygenase and CYP1A1 induction in Hep G2 cells in serum-free culture

The in vitro cellular functions of differentiated cells are influenced by culture conditions. Effects of several extracellular matrices (ECMs) on cytochrome P450-dependent monooxygenases (MFOs) induction and cytochrome P4501A1 (CYP1A1) gene expression were estimated in Hep G2 cells cultured in a serum-free medium. The cells were cultured on collagen type I- and II-, fibronectin-, and matrigel-coated dishes and MFO activities were induced by the addition of 3-methylcholanthrene (MC). The induction of ethoxycoumarin O-deethylase (ECOD) and alkoxyresorufin O-dealkylase activities as well as the expression of CYP1A1 mRNA were also determined. ECOD and methoxy- and ethoxyresorufin O-dealkylase activities in Hep G2 cells were enhanced by culturing the cells using a serum-free medium on fibronectin- or matrigel-coated dishes. ECOD activity on fibronectin-coated dishes was about 3-fold higher than that using a serum-supplemented medium on untreated dishes. Furthermore, both immobilized and soluble fibronectin enhanced the induction of MFOs. The expression of CYP1A1 mRNA using fibronectin-coated dishes was about 2-fold higher than that using a serum-supplemented medium on untreated dishes. These findings suggest that the gene expression in cultured cells is greatly influenced by ECMs. By using fibronectin-coated dishes to cell culture in a serum-free medium, reproducible and highly sensitive results can be obtained in experiments using cultured cells. This revised version was published online in August 2006 with corrections to the Cover Date.     

Cytoskeletal Architecture of Dermal Chromatophores of the Freshwater Teleost Oryzias latipes

Cytoskeletal construction of dermal chromatophores of Orgzias latipes was studied by immunofluorescence microscopy. A microtubule system was most prominent in melanophores where a large number of microtubules emanated from the center of the cell. Xanthophores had an arrangement basically similar to that of melanophores, though the radial pattern became more irregular in the peripheral region where intersecting wavy microtubules were quite frequent. Oval-shaped leucophores exhibited the least-developed microtubule system, where the limited number of microtubules formed a loose basket-like architecture. Intermediate filaments were ubiquitously present in all types of chromatophores and were found to be vimentin-immunoreactive. Examination of doubly-labeled cells indicated that vimentin filaments had similar distribution patterns with microtubules. Orderly arranged bundles of actin filaments were found only in xanthophores, while in melanophores and xanthophores, actin expression was diffuse without displaying a conspicuous filamentous organization. Colchicine treatment induced depolymerization of microtubules and retraction of dendrites in varying degrees in cells in culture and in situ. Melanophores in culture are very sensitive to the treatment while xanthophores appeared to be more resistant in respect to the maintenance of cell morphology.     

Effect of exogenous extracellular matrices on proteoglycan synthesis by cultured rabbit costal chondrocytes

We examined the effect of an extracellular matrix (ECM), produced by either bovine corneal endothelial (BCE) cells or mouse PF HR-9 teratocarcinoma cells, on the ability of rabbit costal chondrocytes to re-express their phenotype once confluent. Rabbit chondrocytes seeded at low densities and grown on plastic tissue culture dishes produced a heterogeneous cell population composed of both overtly differentiated and poorly differentiated chondrocytes, as well as fibroblastic cells. On the other hand, cultures grown on BCE-ECM- or HR-9-ECM-coated dishes reorganized into a homogeneous cartilage-like tissue composed of round cells surrounded by a refractile matrix that stained intensely with alcian green. The cell ultrastructure and that of their pericellular matrix were similar to those seen in vivo. The differentiation of chondrocyte cultures grown on the ECMs vs. plastic was reflected by a two- to three-fold increase in the maximal rate of incorporation of [35S]sulfate and [3H]glucosamine into proteoglycans. Furthermore, the ratio of 35S-labeled proteoglycans incorporated in the cell layer vs. those released into the medium was 1.5-2.5-fold higher when cultures were grown on the ECMs than on plastic. This suggests that the ECMs stimulate the incorporation of newly synthesized proteoglycans into a cartilaginous matrix. Since chondrocyte cultures grown on BCE-ECM or HR-9-ECM give rise to a homogeneous cartilage-like tissue even when seeded at low cell densities, they provide a model for the study of cell-substrate interactions that are responsible for the maintenance of the differentiated phenotype of chondrocytes.     

Serum-free culture of AtT 20 pituitary cells: A system for neuroendocrine studies under defined conditions

Summary The growth of the mouse pituitary cell line AtT 20 was studied under different in vitro conditions. A completely defined, serum-free culture medium supported the survival of cells for a period of more than 2 mo. The medium, designed SFI, consisted of basal medium supplemented with transferrin, insulin, putrescine, and selenium. For maintenance of cells during long-term culture, no additional compounds were necessary. The time-dependent increases in cell number during culture with fetal bovine serum (FBS) and under serum-free conditions showed similar properties. Analysis of the effects of different substrata on cell growth demonstrated that polylysine supported adhesion and initial growth of cells to a greater extent than untreated plastic or FBS adsorbed to culture dishes. Synthesis and regulation of proopiomelanocortin (POMC)-mRNA, the precursor-mRNA of adrenocorticotropin (ACTH), could be detected by Northern blot analysis under basal conditions and after incubation with steroids and corticotropin-releasing hormone (CRH), indicating the serum-independent expression of important cellular properties.     

Pax7 identifies neural crest, chromatophore lineages and pigment stem cells during zebrafish development

Using immunostaining during early zebrafish embryogenesis, we report that the cranial and trunk neural crest expresses the paired box protein Pax7, thus revealing a novel neural crest marker in zebrafish. In the head, we show that Pax7 is broadly expressed in the cranial crest cells, which indicates that duplication of the paralogous group Pax3/7 at the origin of vertebrates included the conserved expression of Pax7 in the head neural crest of all of the vertebrates species studied so far. In the trunk, Pax7 recognizes both premigratory and migratory neural crest cells. Notably, we observed the expression of Pax7 during the development of melanophore, xanthophore and iridophore precursor cells. In contrast to the case of melanocyte precursors in birds, Pax7 showed overlapping expression with early melanin pigment. Finally, during the larva to adult transition, we show that pigment stem cells recapitulate the expression of Pax7.     

Cellular Plasticity Among Axolotl Neural Crest-Derived Pigment Cell Lineages

Many of the factors and mechanisms guiding the migration/differentiation of neural crest cells that give rise to a number of distinguishable cell types, including all dermal and epidermal pigment cells, remain unknown. The axolotl possesses three pigment cell types that differentiate according to specific developmentally programmed sequences and contribute to pigment pattern in the adult. A single lineage of the crest that becomes restricted to one of three pigment cell types gives us the opportunity to examine the existence of a neural crest stem cell population and the potential for transdifferentiation events. Interpretations of experiments involving drug-treated and mutant axolotls implicate cellular plasticity leading to observed phenotypes. We present results from recent in vitro studies designed to identify parameters influencing differentiation events of individual neural crest-derived pigment cell lineages. We demonstrate that the differentiation of xanthophores is enhanced, while that of the melanophores are inhibited in guanosine-supplemented neural crest cell cultures. Data suggest that the increase in one pigment cell population is at the expense of another, indicative of cellular plasticity. Videomicroscopy used in this study agrees with an abundance of correlative evidence supporting the hypothesis of transdifferentiation events among neural crest-derived pigment cell populations. The embryonic neural crest-derived pigment cell system is an ideal model to study differentiation of multipotential stem cells that play critical roles in patterning.