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.     

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.     

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.     

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.     

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.     

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.     

Dynamics of pigment pattern formation in the zebrafish, Brachydanio rerio. III. Effect of anteroposterior location of three-day lateral line melanophores on colonization by the second wave of melanophores

The mode of colonization of the lateral line melanophore band of the zebrafish, Brachydanio rerio, by the second wave of melanophores has been investigated. This stripe forms in two consecutive stages. First, there is an initial migration and reorientation of pigment cells in an anteroposterior wave into the site to form an interrupted stripe. Following this, a round of melanophores differentiates directly at the site and fills in the gaps between the initial cells. An analysis of the distributions of initial and second wave melanophores along the stripe site has shown that both groups of cells are selective as to localization. Initial wave melanophores colonize more anterior somite areas than do second wave melanophores. However, both groups of cells exhibit preferential colonization of the same anterior sites. It is suggested that second wave melanophores attempt to colonize the same somite areas of the stripe as the initial wave of melanophores but are forced to move to more posterior locations due to the presence of initial wave melanophores anteriorly. Observations were also made on later stages of development of the lateral line melanophore band. These melanophores retain the ability to migrate. Some of them reorient out onto the flank and contribute to the juvenile flank pigment pattern.     

Development and evolution of melanophore patterns in fishes of the genus Danio (Teleostei: Cyprinidae)

Pigmentation patterns in vertebrates have become an important model for those interested in mechanisms of pattern determination. I present detailed information on the development of melanophore patterns in the zebrafish, Danio rerio, five close relatives of that species, and an outgroup. The comparison of the ontogeny of melanophore patterns in this group is an important first step towards understanding the developmental basis of the interspecific variation. Pigment patterns in this group range from no distinct patterning at all to stripes of differing numbers and widths to reticulated stripes. Species examined form identical larval patterns and follow a common sequence of events from which different elements are eliminated or altered to produce the variety of patterns seen in the group. As flexion is completed, melanophores move from larval positions onto the flanks of the fish. In D. rerio, D. rerio ‘leo,’ D. kerri, and D. malabaricus, xanthophores become established on the body of the fish as the melanophores move; erythrophores become established on the flanks of D. albolineatus and D. sp. cf. aequipinnatus. An increase in melanophore number, begun at this time, continues at a higher rate in D. rerio, D. kerri, D. sp. cf. aequipinnatus and Tanichthys albonubes than in the other three species. This results in a greater number of melanophores on adults in those species with a higher rate of melanophore increase. No distinct pattern forms, except on the caudal peduncle, in D. albolineatus. In all other Danio species, melanophore stripes form first below then above the horizontal myoseptum. Additional stripes are added first below then above these initial two stripes. D. kerri develops fewer, wider melanophore stripes than D. rerio. After initial stripe formation, D. malabaricus and D. sp. cf. aequipinnatus both developed vertical pattern elements and reticulations in the melanophore pattern. Differences in patterns between species are similar in several cases to described mutants of the zebrafish, suggesting that some aspects of interspecific pigmentation pattern variation may be under relatively simple genetic control. J. Morphol. 241:83–105, 1999. © 1999 Wiley‐Liss, Inc.     

Zebrafish hybrids suggest genetic mechanisms for pigment pattern diversification in Danio

Pigment patterns of Danio fishes are a tractable system for assessing the developmental genetic bases for the evolution of adult form in vertebrates. These pigment patterns include multiple horizontal melanophore stripes in the zebrafish D. rerio, a complete absence of stripes in D. albolineatus, a few broad stripes in D. kerri, and a combination of stripes and spots in D. nigrofasciatus. Here we assess the genetics of pigment pattern development and evolution using interspecific hybrids. We first reconstruct the phylogenetic relationships of these species by analyzing mitochondrial 12S and 16S rDNA sequences. We find a clade comprising several small species of danio, and within this clade a sister taxon relationship between D. rerio and D. nigrofasciatus. We also find that the large bodied D. dangila is more closely related to the clade of small danios than other large bodied species. As a first step in evaluating the genetics of pigment pattern diversification in the group, we then examine the phenotypes of interspecific hybrids. Adult pigment patterns of hybrids between D. rerio and other danios are in many respects more similar to D. rerio than the heterospecific danio, demonstrating that alleles of pigment pattern genes in other species typically are recessive to D. rerio alleles. Furthermore, hybrids between two additional striped species (D. kerri, D. nigrofasciatus) and D. albolineatus suggest that striped patterns are dominant or semi-dominant over an absence of stripes. Together, these analyses support a model in which pigment pattern differences between D. rerio and other species result from gain-of-function alleles in D. rerio, or loss-of-function alleles in other danios. Finally, because several D. rerio pigment pattern mutants resemble heterospecific danios, we use interspecific complementation tests to assess potential roles for these loci in pigment pattern diversification. Crosses between other danios and most D. rerio pigment pattern mutants develop stripes, similar to control hybrids with wild-type D. rerio. These complementation phenotypes allow us to exclude most of these loci as having major effect roles in generating pigment pattern differences between species. In contrast, hybrids between fms mutant D. rerio and D. albolineatus fail to develop stripes, similar to D. albolineatus. This non-complementation phenotype identifies changes in fms, or the pathway in which it acts, as candidates for contributing to the evolutionary loss of stripes in D. albolineatus.     

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

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

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.     

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.     

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.     

Basonuclin-2 Requirements for Zebrafish Adult Pigment Pattern Development and Female Fertility

Relatively little is known about the generation of adult form. One complex adult trait that is particularly amenable to genetic and experimental analysis is the zebrafish pigment pattern, which undergoes extensive remodeling during post-embryonic development to form adult stripes. These stripes result from the arrangement of three classes of neural crest-derived pigment cells, or chromatophores: melanophores, xanthophores, and iridophores. Here, we analyze the zebrafish bonaparte mutant, which has a normal early pigment pattern but exhibits a severe disruption to the adult stripe pattern. We show that the bonaparte mutant phenotype arises from mutations in basonuclin-2 (bnc2), encoding a highly conserved, nuclear-localized zinc finger protein of unknown function. We show that bnc2 acts non-autonomously to the melanophore lineage and is expressed by hypodermal cells adjacent to chromatophores during adult pigment pattern formation. In bonaparte (bnc2) mutants, all three types of chromatophores differentiate but then are lost by extrusion through the skin. We further show that while bnc2 promotes the development of two genetically distinct populations of melanophores in the body stripes, chromatophores of the fins and scales remain unaffected in bonaparte mutants, though a requirement of fin chromatophores for bnc2 is revealed in the absence of kit and colony stimulating factor-1 receptor activity. Finally, we find that bonaparte (bnc2) mutants exhibit dysmorphic ovaries correlating with infertility and bnc2 is expressed in somatic ovarian cells, whereas the related gene, bnc1, is expressed within oocytes; and we find that both bnc2 and bnc1 are expressed abundantly within the central nervous system. These findings identify bnc2 as an important mediator of adult pigment pattern formation and identify bonaparte mutants as an animal model for dissecting bnc2 functions.     

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.     

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.     

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.