Mutational analysis of endothelin receptor b1 (rose) during neural crest and pigment pattern development in the zebrafish Danio rerio

Pigment patterns of fishes are a tractable system for studying the genetic and cellular bases for postembryonic phenotypes. In the zebrafish Danio rerio, neural crest-derived pigment cells generate different pigment patterns during different phases of the life cycle. Whereas early larvae exhibit simple stripes of melanocytes and silver iridophores in a background of yellow xanthophores, this pigment pattern is transformed at metamorphosis into that of the adult, comprising a series of dark melanocyte and iridophore stripes, alternating with light stripes of iridophores and xanthophores. Although several genes have been identified in D. rerio that contribute to the development of both early larval and adult pigment patterns, comparatively little is known about genes that are essential for pattern formation during just one or the other life cycle phase. In this study, we identify the gene responsible for the rose mutant phenotype in D. rerio. rose mutants have wild-type early larval pigment patterns, but fail to develop normal numbers of melanocytes and iridophores during pigment pattern metamorphosis and exhibit a disrupted pattern of these cells. We show that rose corresponds to endothelin receptor b1 (ednrb1), an orthologue of amniote Ednrb genes that have long been studied for their roles in neural crest and pigment cell development. Furthermore, we demonstrate that D. rerio ednrb1 is expressed both during pigment pattern metamorphosis and during embryogenesis, and cells of melanocyte, iridophore, and xanthophore lineages all express this gene. These analyses suggest a phylogenetic conservation of roles for Ednrb signaling in the development of amniote and teleost pigment cell precursors. As murine Ednrb is essential for the development of all neural crest derived melanocytes, and D. rerio ednrb1 is required only by a subset of adult melanocytes and iridophores, these analyses also reveal variation among vertebrates in the cellular requirements for Ednrb signaling, and suggest alternative models for the cellular and genetic bases of pigment pattern metamorphosis in D. rerio.     

Origins of adult pigmentation: diversity in pigment stem cell lineages and implications for pattern evolution

Teleosts comprise about half of all vertebrate species and exhibit an extraordinary diversity of adult pigment patterns that function in shoaling, camouflage, and mate choice and have played important roles in speciation. Here, we review studies that have identified several distinct neural crest lineages, with distinct genetic requirements, that give rise to adult pigment cells in fishes. These lineages include post‐embryonic, peripheral nerve‐associated stem cells that generate black melanophores and iridescent iridophores, cells derived directly from embryonic neural crest cells that generate yellow‐orange xanthophores, and bipotent stem cells that generate both melanophores and xanthophores. This complexity in adult chromatophore lineages has implications for our understanding of adult traits, melanoma, and the evolutionary diversification of pigment cell lineages and patterns.     

Clonal analyses reveal roles of organ founding stem cells, melanocyte stem cells and melanoblasts in establishment, growth and regeneration of the adult zebrafish fin

In vertebrates, the adult form emerges from the embryo by mobilization of precursors or adult stem cells. What different cell types these precursors give rise to, how many precursors establish the tissue or organ, and how they divide to establish and maintain the adult form remain largely unknown. We use the pigment pattern of the adult zebrafish fin, with a variety of clonal and lineage analyses, to address these issues. Early embryonic labeling with lineage-marker-bearing transposons shows that all classes of fin melanocytes (ontogenetic, regeneration and kit-independent melanocytes) and xanthophores arise from the same melanocyte-producing founding stem cells (mFSCs), whereas iridophores arise from distinct precursors. Additionally, these experiments show that, on average, six and nine mFSCs colonize the caudal and anal fin primordia, and daughters of different mFSCs always intercalate to form the adult pattern. Labeled clones are arrayed along the proximal-distal axis of the fin, and melanocyte time-of-differentiation lineage assays show that although most of the pigment pattern growth is at the distal edge of the fin, significant growth also occurs proximally. This suggests that leading edge melanocyte stem cells (MSCs) divide both asymmetrically to generate new melanocytes, and symmetrically to expand the MSCs and leave quiescent MSCs in their wake. Clonal labeling in adult stages confirms this and reveals different contributions of MSCs and transient melanoblasts during growth. These analyses build a comprehensive picture for how MSCs are established and grow to form the pigment stripes of the adult zebrafish fins.     

An orthologue of the kit-related gene fms is required for development of neural crest-derived xanthophores and a subpopulation of adult melanocytes in the zebrafish, Danio rerio

Developmental mechanisms underlying traits expressed in larval and adult vertebrates remain largely unknown. Pigment patterns of fishes provide an opportunity to identify genes and cell behaviors required for postembryonic morphogenesis and differentiation. In the zebrafish, Danio rerio, pigment patterns reflect the spatial arrangements of three classes of neural crest-derived pigment cells: black melanocytes, yellow xanthophores and silver iridophores. We show that the D. rerio pigment pattern mutant panther ablates xanthophores in embryos and adults and has defects in the development of the adult pattern of melanocyte stripes. We find that panther corresponds to an orthologue of the c-fms gene, which encodes a type III receptor tyrosine kinase and is the closest known homologue of the previously identified pigment pattern gene, kit. In mouse, fms is essential for the development of macrophage and osteoclast lineages and has not been implicated in neural crest or pigment cell development. In contrast, our analyses demonstrate that fms is expressed and required by D. rerio xanthophore precursors and that fms promotes the normal patterning of melanocyte death and migration during adult stripe formation. Finally, we show that fms is required for the appearance of a late developing, kit-independent subpopulation of adult melanocytes. These findings reveal an unexpected role for fms in pigment pattern development and demonstrate that parallel neural crest-derived pigment cell populations depend on the activities of two essentially paralogous genes, kit and fms.     

Localization of pigment cells in cultured frog skin

The pigmentation pattern of ventral skin of the frog Rana esculenta consists mainly of melanophores and iridophores, rather than the three pigment cells (xanthophores, iridophores, and melanophores) which form typical dermal chromatophore units in dorsal skin. The present study deals with the precise localization and identification of the types of pigment cells in relation to their position in the dermal tracts of uncultured or cultured frog skins. Iridophores were observed by dark-field microscopy; both melanophores and iridophores were observed by transmission electron microscopy. In uncultured skins, three levels were distinguished in the dermal tracts connecting the subcutaneous tissue to the upper dermis. Melanophores and iridophores were localized in the upper openings of the tracts directed towards the superficial dermis (level 1). The tracts themselves formed level 2 and contained melanophores and a few iridophores. The inner openings of the tracts made up level 3 in which mainly iridophores were present. These latter openings faced the subcutaneous tissue In cultured skins, such pigment-cell distribution remained unchanged, except at level 2 of the tracts, where pigment cells were statistically more numerous; among these, mosaic pigment cells were sometimes observed.     

How fish color their skin: A paradigm for development and evolution of adult patterns

Pigment cells in zebrafish ? melanophores, iridophores, and xanthophores ? originate from neural crest‐derived stem cells associated with the dorsal root ganglia of the peripheral nervous system. Clonal analysis indicates that these progenitors remain multipotent and plastic beyond embryogenesis well into metamorphosis, when the adult color pattern develops. Pigment cells share a lineage with neuronal cells of the peripheral nervous system; progenitors propagate along the spinal nerves. The proliferation of pigment cells is regulated by competitive interactions among cells of the same type. An even spacing involves collective migration and contact inhibition of locomotion of the three cell types distributed in superimposed monolayers in the skin. This mode of coloring the skin is probably common to fish, whereas different patterns emerge by species specific cell interactions among the different pigment cell types. These interactions are mediated by channels involved in direct cell contact between the pigment cells, as well as unknown cues provided by the tissue environment.     

Formation of the adult pigment pattern in zebrafish requires leopard and obelix dependent cell interactions

Colour patterns are a prominent feature of many animals and are of high evolutionary relevance. In zebrafish, the adult pigment pattern comprises alternating stripes of two pigment cell types, melanophores and xanthophores. How the stripes are defined and a straight boundary is formed remains elusive. We find that mutants lacking one pigment cell type lack a striped pattern. Instead, cells of one type form characteristic patterns by homotypic interactions. Using mosaic analysis, we show that juxtaposition of melanophores and xanthophores suffices to restore stripe formation locally. Based on this, we have analysed the pigment pattern of two adult specific mutants: leopard and obelix. We demonstrate that obelix is required in melanophores to promote their aggregation and controls boundary integrity. By contrast, leopard regulates homotypic interaction within both melanophores and xanthophores, and interaction between the two, thus controlling boundary shape. These findings support a view in which cell-cell interactions among pigment cells are the major driving force for adult pigment pattern formation.     

pyewacket, a new zebrafish fin pigment pattern mutant

Many mutants that disrupt zebrafish embryonic pigment pattern have been isolated, and subsequent cloning of the mutated genes causing these phenotypes has contributed to our understanding of pigment cell development. However, few mutants have been identified that specifically affect development of the adult pigment pattern. Through a mutant screen for adult pigment pattern phenotypes, we identified pyewacket (pye), a novel zebrafish mutant in which development of the adult caudal fin pigment pattern is aberrant. Specifically, pye mutants have fin melanocyte pigment pattern defects and fewer xanthophores than wild-type fins. We mapped pye to an interval where a single gene, the zebrafish ortholog of the human gene DHRSX, is present. pye will be an informative mutant for understanding how xanthophores and melanocytes interact to form the pigment pattern of the adult zebrafish fin.     

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制提供了基础资料。     

Pigment cell mechanisms underlying dorsal color‐pattern polymorphism in the japanese four‐lined snake

To provide histological foundation for studying the genetic mechanisms of color‐pattern polymorphisms, we examined light reflectance profiles and cellular architectures of pigment cells that produced striped, nonstriped, and melanistic color patterns in the snake Elaphe quadrivirgata. Both, striped and nonstriped morphs, possessed the same set of epidermal melanophores and three types of dermal pigment cells (yellow xanthophores, iridescent iridophores, and black melanophores), but spatial variations in the densities of epidermal and dermal melanophores produced individual variations in stripe vividness. The densities of epidermal and dermal melanophores were two or three times higher in the dark‐brown‐stripe region than in the yellow background in the striped morph. However, the densities of epidermal and dermal melanophores between the striped and background regions were similar in the nonstriped morph. The melanistic morph had only epidermal and dermal melanophores and neither xanthophores nor iridophores were detected. Ghost stripes in the shed skin of some melanistic morphs suggested that stripe pattern formation and melanism were controlled independently. We proposed complete‐ and incomplete‐dominance heredity models for the stripe‐melanistic variation and striped, pale‐striped, and nonstriped polymorphisms, respectively, according to the differences in pigment‐cell composition and its spatial architecture. J. Morphol. 274:1353–1364, 2013. © 2013 Wiley Periodicals, Inc.     

The influence of long-term chromatic adaptation on pigment cells and striped pigment patterns in the skin of the zebrafish, Danio rerio

The striped pigment patterns in the flanks of zebrafish result from chromatophores deep within the dermis or hypodermis, while superficial melanophores associated with dermal scales add a dark tint to the dorsal coloration. The responses of these chromatophores were compared during the long-term adaptation of zebrafish to a white or a black background. In superficial skin, melanophores, xanthophores, and two types of iridophores are distributed in a gradient along the dorso-ventral axis independent of the hypodermal pigment patterns. Within one week the superficial melanophores and iridophores changed their density and/or areas of distribution, which adopted the dorsal skin color and the hue of the flank to the background, but did not affect the striped pattern. The increases or decreases in superficial melanophores are thought to be caused by apoptosis or by differentiation, respectively. When the adaptation period was prolonged for more than several months, the striped color pattern was also affected by changes in the width of the black stripes. Some black stripes disappeared and interstripe areas were emphasized with a yellow color within one year on a white background. Such long-term alteration in the pigment pattern was caused by a decrease in the distribution of melanophores and a concomitant increase in xanthophores in the hypodermis. These results indicate that morphological responses of superficial chromatophores contribute to the effective and rapid background adaptation of dorsal skin and while prolonged adaptation also affects hypodermal chromatophores in the flank to alter the striped pigment patterns.     

Sox5 Functions as a Fate Switch in Medaka Pigment Cell Development

Mechanisms generating diverse cell types from multipotent progenitors are crucial for normal development. Neural crest cells (NCCs) are multipotent stem cells that give rise to numerous cell-types, including pigment cells. Medaka has four types of NCC-derived pigment cells (xanthophores, leucophores, melanophores and iridophores), making medaka pigment cell development an excellent model for studying the mechanisms controlling specification of distinct cell types from a multipotent progenitor. Medaka many leucophores-3 (ml-3) mutant embryos exhibit a unique phenotype characterized by excessive formation of leucophores and absence of xanthophores. We show that ml-3 encodes sox5, which is expressed in premigratory NCCs and differentiating xanthophores. Cell transplantation studies reveal a cell-autonomous role of sox5 in the xanthophore lineage. pax7a is expressed in NCCs and required for both xanthophore and leucophore lineages; we demonstrate that Sox5 functions downstream of Pax7a. We propose a model in which multipotent NCCs first give rise to pax7a-positive partially fate-restricted intermediate progenitors for xanthophores and leucophores; some of these progenitors then express sox5, and as a result of Sox5 action develop into xanthophores. Our results provide the first demonstration that Sox5 can function as a molecular switch driving specification of a specific cell-fate (xanthophore) from a partially-restricted, but still multipotent, progenitor (the shared xanthophore-leucophore progenitor).     

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.     

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.     

Extracellular Matrix Constituents and Pigment Cell Expression in Primary Cell Culture

Three types of pigment cells were isolated and cultured from larval Rana pipiens, and their attachment, maintenance, and proliferation were examined in the presence of extra-cellular matrix constituents (ECMs) in primary cell culture. The initial profile of pigment cell types present on day 2 of culture reflects the relative attachment of the cells to the dishes. Changes in the numbers of cells present after day 2 reflects the influence of factors present in the culture media on the maintenance, proliferation, or detachment of each type of pigment cell. Fetal bovine serum (FBS) promoted melanophore expression, but inhibited iridophore expression. FBS had no effect on xanthophores. In contrast, ventral skin conditioned medium (VCM), which contains melanization inhibiting factor, strongly stimulated iridophore expression, while it markedly inhibited melanophore expression. VCM had little effect on xanthophores. Of the ECMs tested, collagen type I had no effect on pigment cells. Fibronectin slightly inhibited melanophore expression, while it moderately stimulated iridophores and xanthophores. The stimulatory effect of fibronectin was not as strong as that of FBS or VCM. Laminin was also tested; however, it did not allow pigment cells to attach to the dishes, at least under the culture conditions utilized. The results of these experiments are discussed in terms of the general mechanisms of pigment pattern formation.     

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.     

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.     

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.