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.     

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.     

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.     

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.     

Deconstructing evolution of adult phenotypes: genetic analyses of kit reveal homology and evolutionary novelty during adult pigment pattern development of Danio fishes

The cellular bases for evolutionary changes in adult form remain largely unknown. Pigment patterns of Danio fishes are a convenient system for studying these issues because of their diversity and accessibility and because one species, the zebrafish D. rerio, is a model organism for biomedical research. Previous studies have shown that in zebrafish, stripes form by migration and differentiation of distinct populations of melanophores: early metamorphic (EM) melanophores arise widely dispersed and then migrate into stripes, whereas late metamorphic (LM) melanophores arise already within stripes. EM melanophores require the kit receptor tyrosine kinase, as kit mutants lack these cells but retain LM melanophores, which form a residual stripe pattern. To see if similar cell populations and genetic requirements are present in other species, we examined D. albolineatus, which has relatively few, nearly uniform melanophores. We isolated a D. albolineatus kit mutant and asked whether residual, LM melanophores develop in this species, as in D. rerio. We found that kit mutant D. albolineatus lack EM melanophores, yet retain LM melanophores. Histological analyses further show that kit functions during a late step in metamorphic melanophore development in both species. Interestingly, kit mutant D. albolineatus develop a striped melanophore pattern similar to kit mutant D. rerio, revealing latent stripe-forming potential in this species, despite its normally uniform pattern. Comparisons of wild types and kit mutants of the two species further show that species differences in pigment pattern reflect: (1) changes in the behavior of kit-dependent EM melanophores that arise in a dispersed pattern and then migrate into stripes in D. rerio, but fail to migrate in D. albolineatus; and (2) a change in the number of kit-independent LM melanophores that arise already in stripes and are numerous in D. rerio, but few in D. albolineatus. Our results show how genetic analyses of a species closely related to a biomedical model organism can reveal both conservatism and innovation in developmental mechanisms underlying evolutionary changes in adult form.     

Pigment pattern in jaguar/obelix zebrafish is caused by a Kir7.1 mutation: implications for the regulation of melanosome movement

Many animals have a variety of pigment patterns, even within a species, and these patterns may be one of the driving forces of speciation. Recent molecular genetic studies on zebrafish have revealed that interaction among pigment cells plays a key role in pattern formation, but the mechanism of pattern formation is unclear. The zebrafish jaguar/obelix mutant has broader stripes than wild-type fish. In this mutant, the development of pigment cells is normal but their distribution is altered, making these fish ideal for studying the process of pigment pattern formation. Here, we utilized a positional cloning method to determine that the inwardly rectifying potassium channel 7.1 (Kir7.1) gene is responsible for pigment cell distribution among jaguar/obelix mutant fish. Furthermore, in jaguar/obelix mutant alleles, we identified amino acid changes in the conserved region of Kir7.1, each of which affected K⁺ channel activity as demonstrated by patch-clamp experiments. Injection of a bacterial artificial chromosome containing the wild-type Kir7.1 genomic sequence rescued the jaguar/obelix phenotype. From these results, we conclude that mutations in Kir7.1 are responsible for jaguar/obelix. We also determined that the ion channel function defect of melanophores expressing mutant Kir7.1 altered the cellular response to external signals. We discovered that mutant melanophores cannot respond correctly to the melanosome dispersion signal derived from the sympathetic neuron and that melanosome aggregation is constitutively activated. In zebrafish and medaka, it is well known that melanosome aggregation and subsequent melanophore death increase when fish are kept under constant light conditions. These observations indicate that melanophores of jaguar/obelix mutant fish have a defect in the signaling pathway downstream of the α₂-adrenoceptor. Taken together, our results suggest that the cellular defect of the Kir7.1 mutation is directly responsible for the pattern change in the jaguar/obelix mutant.     

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.     

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.     

Embryonic requirements for ErbB signaling in neural crest development and adult pigment pattern formation

Vertebrate pigment cells are derived from neural crest cells and are a useful system for studying neural crest-derived traits during post-embryonic development. In zebrafish, neural crest-derived melanophores differentiate during embryogenesis to produce stripes in the early larva. Dramatic changes to the pigment pattern occur subsequently during the larva-to-adult transformation, or metamorphosis. At this time, embryonic melanophores are replaced by newly differentiating metamorphic melanophores that form the adult stripes. Mutants with normal embryonic/early larval pigment patterns but defective adult patterns identify factors required uniquely to establish, maintain or recruit the latent precursors to metamorphic melanophores. We show that one such mutant, picasso, lacks most metamorphic melanophores and results from mutations in the ErbB gene erbb3b, which encodes an EGFR-like receptor tyrosine kinase. To identify critical periods for ErbB activities, we treated fish with pharmacological ErbB inhibitors and also knocked down erbb3b by morpholino injection. These analyses reveal an embryonic critical period for ErbB signaling in promoting later pigment pattern metamorphosis, despite the normal patterning of embryonic/early larval melanophores. We further demonstrate a peak requirement during neural crest migration that correlates with early defects in neural crest pathfinding and peripheral ganglion formation. Finally, we show that erbb3b activities are both autonomous and non-autonomous to the metamorphic melanophore lineage. These data identify a very early, embryonic, requirement for erbb3b in the development of much later metamorphic melanophores, and suggest complex modes by which ErbB signals promote adult pigment pattern development.     

Melanophore Migration and Survival during Zebrafish Adult Pigment Stripe Development Require the Immunoglobulin Superfamily Adhesion Molecule Igsf11

The zebrafish adult pigment pattern has emerged as a useful model for understanding the development and evolution of adult form as well as pattern-forming mechanisms more generally. In this species, a series of horizontal melanophore stripes arises during the larval-to-adult transformation, but the genetic and cellular bases for stripe formation remain largely unknown. Here, we show that the seurat mutant phenotype, consisting of an irregular spotted pattern, arises from lesions in the gene encoding Immunoglobulin superfamily member 11 (Igsf11). We find that Igsf11 is expressed by melanophores and their precursors, and we demonstrate by cell transplantation and genetic rescue that igsf11 functions autonomously to this lineage in promoting adult stripe development. Further analyses of cell behaviors in vitro, in vivo, and in explant cultures ex vivo demonstrate that Igsf11 mediates adhesive interactions and that mutants for igsf11 exhibit defects in both the migration and survival of melanophores and their precursors. These findings identify the first in vivo requirements for igsf11 as well as the first instance of an immunoglobulin superfamily member functioning in pigment cell development and patterning. Our results provide new insights into adult pigment pattern morphogenesis and how cellular interactions mediate pattern formation.     

Zebrafish endzone regulates neural crest-derived chromatophore differentiation and morphology

The development of neural crest-derived pigment cells has been studied extensively as a model for cellular differentiation, disease and environmental adaptation. Neural crest-derived chromatophores in the zebrafish (Danio rerio) consist of three types: melanophores, xanthophores and iridiphores. We have identified the zebrafish mutant endzone (enz), that was isolated in a screen for mutants with neural crest development phenotypes, based on an abnormal melanophore pattern. We have found that although wild-type numbers of chromatophore precursors are generated in the first day of development and migrate normally in enz mutants, the numbers of all three chromatophore cell types that ultimately develop are reduced. Further, differentiated melanophores and xanthophores subsequently lose dendricity, and iridiphores are reduced in size. We demonstrate that enz function is required cell autonomously by melanophores and that the enz locus is located on chromosome 7. In addition, zebrafish enz appears to selectively regulate chromatophore development within the neural crest lineage since all other major derivatives develop normally. Our results suggest that enz is required relatively late in the development of all three embryonic chromatophore types and is normally necessary for terminal differentiation and the maintenance of cell size and morphology. Thus, although developmental regulation of different chromatophore sublineages in zebrafish is in part genetically distinct, enz provides an example of a common regulator of neural crest-derived chromatophore differentiation and morphology.     

Melanophores in the stripes of adult zebrafish do not have the nature to gather,but disperse when they have the space to move

Animal skin pattern is one of the good model systems used to study the mechanism of pattern formation. Molecular genetic studies with zebrafish have shown that pigment cells play a major role in the mechanism of stripe formation. Among the variety of cellular events that may be involved in the mechanism, aggregation of melanophores has been suggested as an important factor for pattern formation. However, only a few experimental studies detected the migration ability of melanophores in vivo. Here, we tried to determine whether melanophores really have the ability to aggregate in the skin of zebrafish. Melanophores in the adult stripes are packed densely and they rarely move. However, when the neighboring pigment cells are killed, they move and regenerate the stripe pattern, suggesting that melanophores retain the migration ability. To analyze the migration, we ablated a part of the melanophores by laser to give free space to the remaining cells; we then traced the migration. Contrary to our expectation, we found that melanophores repulsed one another and dispersed from the aggregated condition in the absence of xanthophores. Apparent aggregation may be forced by the stronger repulsive effect against the xanthophores, which excludes melanophores from the yellow stripe region.     

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.     

oca2 regulation of chromatophore differentiation and number is cell type specific in zebrafish

We characterized a zebrafish mutant that displays defects in melanin synthesis and in the differentiation of melanophores and iridophores of the skin and retinal pigment epithelium. Positional cloning and candidate gene sequencing link this mutation to a 410‐kb region on chromosome 6, containing the oculocutaneous albinism 2 (oca2) gene. Quantification of oca2 mutant melanophores shows a reduction in the number of differentiated melanophores compared with wildtype siblings. Consistent with the analysis of mouse Oca2‐deficient melanocytes, zebrafish mutant melanophores have immature melanosomes which are partially rescued following treatment with vacuolar‐type ATPase inhibitor/cytoplasmic pH modifier, bafilomycin A1. Melanophore‐specific gene expression is detected at the correct time and in anticipated locations. While oca2 zebrafish display unpigmented gaps on the head region of mutants 3 days post‐fertilization, melanoblast quantification indicates that oca2 mutants have the correct number of melanoblasts, suggesting a differentiation defect explains the reduced melanophore number. Unlike melanophores, which are reduced in number in oca2 mutants, differentiated iridophores are present at significantly higher numbers. These data suggest distinct mechanisms for oca2 in establishing differentiated chromatophore number in developing zebrafish.     

Pigment pattern evolution by differential deployment of neural crest and post-embryonic melanophore lineages in Danio fishes

Latent precursors or stem cells of neural crest origin are present in a variety of post-embryonic tissues. Although these cells are of biomedical interest for roles in human health and disease, their potential evolutionary significance has been underappreciated. As a first step towards elucidating the contributions of such cells to the evolution of vertebrate form, we investigated the relative roles of neural crest cells and post-embryonic latent precursors during the evolutionary diversification of adult pigment patterns in Danio fishes. These pigment patterns result from the numbers and arrangements of embryonic melanophores that are derived from embryonic neural crest cells, as well as from post-embryonic metamorphic melanophores that are derived from latent precursors of presumptive neural crest origin. In the zebrafish D. rerio, a pattern of melanophore stripes arises during the larval-to-adult transformation by the recruitment of metamorphic melanophores from latent precursors. Using a comparative approach in the context of new phylogenetic data, we show that adult pigment patterns in five additional species also arise from metamorphic melanophores, identifying this as an ancestral mode of adult pigment pattern development. By contrast, superficially similar adult stripes of D. nigrofasciatus (a sister species to D. rerio) arise by the reorganization of melanophores that differentiated at embryonic stages, with a diminished contribution from metamorphic melanophores. Genetic mosaic and molecular marker analyses reveal evolutionary changes that are extrinsic to D. nigrofasciatus melanophore lineages, including a dramatic reduction of metamorphic melanophore precursors. Finally, interspecific complementation tests identify a candidate genetic pathway for contributing to the evolutionary reduction in metamorphic melanophores and the increased contribution of early larval melanophores to D. nigrofasciatus adult pigment pattern development. These results demonstrate an important role for latent precursors in the diversification of pigment patterns across danios. More generally, differences in the deployment of post-embryonic neural crest-derived stem cells or their specified progeny may contribute substantially to the evolutionary diversification of adult form in vertebrates, particularly in species that undergo a metamorphosis.     

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.     

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.     

Post-embryonic nerve-associated precursors to adult pigment cells: genetic requirements and dynamics of morphogenesis and differentiation

The pigment cells of vertebrates serve a variety of functions and generate a stunning variety of patterns. These cells are also implicated in human pathologies including melanoma. Whereas the events of pigment cell development have been studied extensively in the embryo, much less is known about morphogenesis and differentiation of these cells during post-embryonic stages. Previous studies of zebrafish revealed genetically distinct populations of embryonic and adult melanophores, the ectotherm homologue of amniote melanocytes. Here, we use molecular markers, vital labeling, time-lapse imaging, mutational analyses, and transgenesis to identify peripheral nerves as a niche for precursors to adult melanophores that subsequently migrate to the skin to form the adult pigment pattern. We further identify genetic requirements for establishing, maintaining, and recruiting precursors to the adult melanophore lineage and demonstrate novel compensatory behaviors during pattern regulation in mutant backgrounds. Finally, we show that distinct populations of latent precursors having differential regenerative capabilities persist into the adult. These findings provide a foundation for future studies of post-embryonic pigment cell precursors in development, evolution, and neoplasia.     

Observations on the ultrastructure and distribution of chromatophores in the skin of chelonians

Alibardi, L. 2011. Observations on the ultrastructure and distribution of chromatophores in the skin of chelonians. —Acta Zoologica (Stockholm) 00 :1–11. The cytology and distribution of chromatophores responsible for skin pigmentation in chelonians is analyzed. Epidermal melanocytes are involved in the formation of dark spots or stripes in growing shelled and non‐shelled skin. Melanocytes rest in the basal layer of the epidermis and transfer melanosomes into keratinocytes during epidermal growth. Dermal melanophores and other chromatophores instead remain in the dermis and form the gray background of the skin. When dermal melanophores condense, they give origin to the dense spots or stripes in areas where no epidermal melanocytes are present. In the latter case, the epidermis and the corneous layer are transparent and reveal the dermal distribution of melanophores and other chromatophores underneath. As a result of this basic process of distribution of pigment cells, the dark areas visible in scales can have a double origin (epidermal and dermal) or a single origin (epidermal or dermal). Xanthophores, lipophores, and a cell containing both pterinosomes and lipid droplets are sparse in the loose dermis while iridophores are rarely seen in the skin of chelonians analyzed in the present study. Xanthophores and lipophores contribute to form the pale, yellow or oranges hues present among the dark areas of the skin in turtles.