Ajeet Pratap Singh

Proliferation, dispersal and patterned aggregation of iridophores in the skin prefigure striped colouration of zebrafish

Colour patterns are a striking feature of animals; they evolve rapidly and play an important role in natural as well as sexual selection. It has been proposed that colour pattern formation in adult vertebrates depends on Turing-type interactions between pigment cells; however, little is known about the actual developmental mechanisms underlying the complex and prolonged ontogeny of this important adult feature. Zebrafish (Danio rerio) owe their name to a repetitive pattern of dark stripes and light interstripes parallel to the anteroposterior body axis that develop during juvenile stages. By inducible Cre/loxP-mediated recombination in neural-crest-derived progenitors, we created labelled clones of skin pigment cells that were imaged over several weeks in juvenile and adult fish. Metamorphic iridophores arise from postembryonic stem cells located at the dorsal root ganglia of the peripheral nervous system. They emerge in the skin at the horizontal myoseptum to form the first interstripe and proliferate while spreading bidirectionally along the dorsoventral axis. Patterned aggregation of iridophores during their dispersal generates a series of interstripes that define the stripe regions. Melanophore progenitors appear in situ in the presumptive stripe region where they melanize and expand in size to form compact stripes. Thus, although depending on mutual interactions between different pigment cells, stripes and interstripes are formed by a completely different cellular route.

Zebrafish Stripes as a Model for Vertebrate Colour Pattern Formation

Colour patterns are prominent features of many animals and have important functions in communication, such as camouflage, kin recognition and mate choice. As targets for natural as well as sexual selection, they are of high evolutionary significance. The molecular mechanisms underlying colour pattern formation in vertebrates are not well understood. Progress in transgenic tools, in vivo imaging and the availability of a large collection of mutants make the zebrafish (Danio rerio) an attractive model to study vertebrate colouration. Zebrafish display golden and blue horizontal stripes that form during metamorphosis as mosaics of yellow xanthophores, silvery or blue iridophores and black melanophores in the hypodermis. Lineage tracing revealed the origin of the adult pigment cells and their individual cellular behaviours during the formation of the striped pattern. Mutant analysis indicated that interactions between all three pigment cell types are required for the formation of the pattern, and a number of cell surface molecules and signalling systems have been identified as mediators of these interactions. The understanding of the mechanisms that underlie colour pattern formation is an important step towards deciphering the genetic basis of variation in evolution.

Local reorganization of xanthophores fine-tunes and colors the striped pattern of zebrafish

Origin of fish pigment cell for pattern Zebrafish stripes arise from the interactions of pigment cells: black melanophores, iridescent iridophores, and yellow-orange xanthophores. Melanophores and iridophores develop from nerve-associated stem cells, but the origin of xanthophores is unclear. Two studies now reveal that adult xanthophores originate from xanthophores in embryonic and larval fish, when they proliferate to cover the skin before the arrival of black and silver cells in a striped arrangement. Mahalwar et al. show that xanthophores change their final shape and color depending on their location. In black cells, xanthophores appear faint and stellate, but in silver cells, they are bright and compact. Precise superposition creates the blue and golden colors. McMenamin et al. observe the loss of pigment in embryonic xanthophores and the later reappearance in the adult. They show that redifferentiation depends on the thyroid hormone that also limits melanophore population expansion. Science, this issue p. 1362 and p. 1358 Adult fish pigment xanthophores originate from larval xanthophores and acquire different shapes over stripes. The pattern of alternating blue and golden stripes displayed by adult zebrafish is composed of three kinds of pigment cells: black melanophores, yellow xanthophores, and silvery-blue iridophores. We analyzed the dynamics of xanthophores during stripe morphogenesis in vivo with long-term time-lapse imaging. Larval xanthophores start to proliferate at the onset of metamorphosis and give rise to adult xanthophores covering the flank before the arrival of stem-cell–derived iridophores and melanophores. Xanthophores compact to densely cover the iridophores forming the interstripe, and they acquire a loose stellate shape over the melanophores in the stripes. Thus, xanthophores, attracted by iridophores and repelling melanophores, sharpen and color the pattern. Variations on these cell behaviors may contribute to the generation of color pattern diversity in fish.

Genetic lineage labeling in zebrafish uncovers novel neural crest contributions to the head, including gill pillar cells

At the protochordate-vertebrate transition, a new predatory lifestyle and increased body size coincided with the appearance of a true head. Characteristic innovations of this head are a skull protecting and accommodating a centralized nervous system, a jaw for prey capture and gills as respiratory organs. The neural crest (NC) is a major ontogenetic source for the ‘new head’ of vertebrates and its contribution to the cranial skeleton has been intensively studied in different model organisms. However, the role of NC in the expansion of the respiratory surface of the gills has been neglected. Here, we use genetic lineage labeling to address the contribution of NC to specific head structures, in particular to the gills of adult zebrafish. We generated a sox10:ERT2-Cre line and labeled NC cells by inducing Cre/loxP recombination with tamoxifen at embryonic stages. In juvenile and adult fish, we identified numerous established NC derivatives and, in the cranium, we precisely defined the crest/mesoderm interface of the skull roof. We show the NC origin of the opercular bones and of multiple cell types contributing to the barbels, chemosensory organs located in the mouth region. In the gills, we observed labeled primary and secondary lamellae. Clonal analysis reveals that pillar cells, a craniate innovation that mechanically supports the filaments and forms gill-specific capillaries, have a NC origin. Our data point to a crucial role for the NC in enabling more efficient gas exchange, thus uncovering a novel, direct involvement of this embryonic tissue in the evolution of respiratory systems at the protochordate-vertebrate transition.

The Developmental Genetics of Vertebrate Color Pattern Formation: Lessons from Zebrafish.

Color patterns are prominent features of many animals; they are highly variable and evolve rapidly leading to large diversities even within a single genus. As targets for natural as well as sexual selection, they are of high evolutionary significance. The zebrafish (Danio rerio) has become an important model organism for developmental biology and biomedical research in general, and it is the model organism to study color pattern formation in vertebrates. The fish display a conspicuous pattern of alternating blue and golden stripes on the body and on the anal and tail fins. This pattern is produced by three different types of pigment cells (chromatophores) arranged in precise layers in the hypodermis of the fish. In this essay, we will summarize the recent advances in understanding the developmental and genetic basis for stripe formation in the zebrafish. We will describe the cellular events leading to the formation of stripes during metamorphosis based on long-term lineage imaging. Mutant analysis has revealed that a number of signaling pathways are involved in the establishment and maintenance of the individual pigment cells. However, the striped pattern itself is generated by self-organizing mechanisms requiring interactions between all three pigment cell types. The involvement of integral membrane proteins, including connexins and potassium channels, suggests that direct physical contacts between chromatophores are involved, and that the directed transport of small molecules or bioelectrical coupling is important for these interactions. This mode of patterning by transmitting spatial information between adjacent tissues within three superimposed cell layers is unprecedented in other developmental systems. We propose that variations in the patterns among Danio species are caused by allelic differences in the genes responsible for these interactions.

The Developmental Genetics of Vertebrate Color Pattern Formation

Color patterns are prominent features of many animals; they are highly variable and evolve rapidly leading to large diversities even within a single genus. As targets for natural as well as sexual selection, they are of high evolutionary significance. The zebrafish (Danio rerio) has become an important model organism for developmental biology and biomedical research in general, and it is the model organism to study color pattern formation in vertebrates. The fish display a conspicuous pattern of alternating blue and golden stripes on the body and on the anal and tail fins. This pattern is produced by three different types of pigment cells (chromatophores) arranged in precise layers in the hypodermis of the fish. In this essay, we will summarize the recent advances in understanding the developmental and genetic basis for stripe formation in the zebrafish. We will describe the cellular events leading to the formation of stripes during metamorphosis based on long-term lineage imaging. Mutant analysis has revealed that a number of signaling pathways are involved in the establishment and maintenance of the individual pigment cells. However, the striped pattern itself is generated by self-organizing mechanisms requiring interactions between all three pigment cell types. The involvement of integral membrane proteins, including connexins and potassium channels, suggests that direct physical contacts between chromatophores are involved, and that the directed transport of small molecules or bioelectrical coupling is important for these interactions. This mode of patterning by transmitting spatial information between adjacent tissues within three superimposed cell layers is unprecedented in other developmental systems. We propose that variations in the patterns among Danio species are caused by allelic differences in the genes responsible for these interactions.

Homotypic cell competition regulates proliferation and tiling of zebrafish pigment cells during colour pattern formation

The adult striped pattern of zebrafish is composed of melanophores, iridophores and xanthophores arranged in superimposed layers in the skin. Previous studies have revealed that the assembly of pigment cells into stripes involves heterotypic interactions between all three chromatophore types. Here we investigate the role of homotypic interactions between cells of the same chromatophore type. Introduction of labelled progenitors into mutants lacking the corresponding cell type allowed us to define the impact of competitive interactions via long-term in vivo imaging. In the absence of endogenous cells, transplanted iridophores and xanthophores show an increased rate of proliferation and spread as a coherent net into vacant space. By contrast, melanophores have a limited capacity to spread in the skin even in the absence of competing endogenous cells. Our study reveals a key role for homotypic competitive interactions in determining number, direction of migration and individual spacing of cells within chromatophore populations.

Zebrafish Leucocyte tyrosine kinase controls iridophore establishment, proliferation and survival.

The zebrafish striped pattern results from the interplay among three pigment cell types; black melanophores, yellow xanthophores and silvery iridophores, making it a valuable model to study pattern formation in vivo. It has been suggested that iridophore proliferation, dispersal and cell shape transitions play an important role during stripe formation; however, the underlying molecular mechanisms remain poorly understood. Using gain‐ and loss‐of‐function alleles of leucocyte tyrosine kinase (ltk) and a pharmacological inhibitor approach, we show that Ltk specifically regulates iridophore establishment, proliferation and survival. Mutants in shady/ltk lack iridophores and display an abnormal body stripe pattern. Moonstone mutants, ltkmne, display ectopic iridophores, suggesting hyperactivity of the mutant Ltk. The dominant ltkmne allele carries a missense mutation in a conserved position of the kinase domain that highly correlates with neuroblastomas in mammals. Chimeric analysis suggests a novel physiological role of Ltk in the regulation of iridophore proliferation by homotypic competition.

Response to Comment on"Local reorganization of xanthophores fine-tunes and colors the striped pattern of zebrafish"

Watanabe and Kondo question our conclusion that the current Turing-type model of color patterning in zebrafish requires modification. In addition to xanthophores and melanophores, iridophores are essential for stripe formation in the body, although not in the fins. A model of predictive value should accommodate the in vivo dynamics and interactions of all three chromatophore types in body stripe formation.

Heterotypic interactions regulate cell shape and density during color pattern formation in zebrafish

ABSTRACT The conspicuous striped coloration of zebrafish is produced by cell-cell interactions among three different types of chromatophores: black melanophores, orange/yellow xanthophores and silvery/blue iridophores. During color pattern formation xanthophores undergo dramatic cell shape transitions and acquire different densities, leading to compact and orange xanthophores at high density in the light stripes, and stellate, faintly pigmented xanthophores at low density in the dark stripes. Here, we investigate the mechanistic basis of these cell behaviors in vivo, and show that local, heterotypic interactions with dense iridophores regulate xanthophore cell shape transition and density. Genetic analysis reveals a cell-autonomous requirement of gap junctions composed of Cx41.8 and Cx39.4 in xanthophores for their iridophore-dependent cell shape transition and increase in density in light-stripe regions. Initial melanophore-xanthophore interactions are independent of these gap junctions; however, subsequently they are also required to induce the acquisition of stellate shapes in xanthophores of the dark stripes. In summary, we conclude that, whereas homotypic interactions regulate xanthophore coverage in the skin, their cell shape transitions and density is regulated by gap junction-mediated, heterotypic interactions with iridophores and melanophores. Summary: The conspicuous pigmentation pattern of zebrafish is produced by three kinds of interacting pigment cells. Here we address the cellular consequences of these interactions in wild-type fish and mutants with altered pigment patterns.