Zebrafish Pigment Pattern Formation: Insights into the Development and Evolution of Adult Form.

Vertebrate pigment patterns are diverse and fascinating adult traits that allow animals to recognize conspecifics, attract mates, and avoid predators. Pigment patterns in fish are among the most amenable traits for studying the cellular basis of adult form, as the cells that produce diverse patterns are readily visible in the skin during development. The genetic basis of pigment pattern development has been most studied in the zebrafish, Danio rerio. Zebrafish adults have alternating dark and light horizontal stripes, resulting from the precise arrangement of three main classes of pigment cells: black melanophores, yellow xanthophores, and iridescent iridophores. The coordination of adult pigment cell lineage specification and differentiation with specific cellular interactions and morphogenetic behaviors is necessary for stripe development. Besides providing a nice example of pattern formation responsible for an adult trait of zebrafish, stripe-forming mechanisms also provide a conceptual framework for posing testable hypotheses about pattern diversification more broadly. Here, we summarize what is known about lineages and molecular interactions required for pattern formation in zebrafish, we review some of what is known about pattern diversification in Danio, and we speculate on how patterns in more distant teleosts may have evolved to produce a stunningly diverse array of patterns in nature.

The dynamic behavior of chromatophores marks the transition from bands to spots in leopard geckos.

Reptilian skin coloration is spectacular and diverse, yet little is known about the ontogenetic processes that govern its establishment and the molecular signaling pathways that determine it. Here, we focus on the development of the banded pattern of leopard gecko hatchlings and the transition to black spots in the adult. With our histological analyses, we show that iridophores are present in the white and yellow bands of the hatchling and they gradually perish in the adult skin. Furthermore, we demonstrate that melanophores can autonomously form spots in the absence of the other chromatophores both on the regenerated skin of the tail and on the dorsal skin of the Mack Super Snow (MSS) leopard geckos. This color morph is characterized by uniform black coloration in hatchlings and black spots in adulthood; we establish that their skin is devoid of xanthophores and iridophores at both stages. Our genetic analyses identified a 13-nucleotide deletion in the PAX7 transcription factor of MSS geckos, affecting its protein coding sequence. With our single-cell transcriptomics analysis of embryonic skin, we confirm that PAX7 is expressed in iridophores and xanthophores, suggesting that it plays a key role in the differentiation of both chromatophores. Our in situ hybridizations on whole-mount embryos document the dynamics of the skin pattern formation and how it is impacted in the PAX7 mutants. We hypothesize that the melanophores-iridophores interactions give rise to the banded pattern of the hatchlings and black spot formation is an intrinsic capacity of melanophores in the postembryonic skin.

kcnj13 regulates pigment cell shapes in zebrafish and has diverged by cis-regulatory evolution between Danio species.

Teleost fish of the genus Danio are excellent models to study the genetic and cellular bases of pigment pattern variation in vertebrates. The two sister species Danio rerio and Danio aesculapii show divergent patterns of horizontal stripes and vertical bars that are partly caused by the divergence of the potassium channel gene kcnj13. Here, we show that kcnj13 is required only in melanophores for interactions with xanthophores and iridophores, which cause location-specific pigment cell shapes and thereby influence colour pattern and contrast in D. rerio. Cis-regulatory rather than protein coding changes underlie kcnj13 divergence between the two Danio species. Our results suggest that homotypic and heterotypic interactions between the pigment cells and their shapes diverged between species by quantitative changes in kcnj13 expression during pigment pattern diversification.

The genetic basis of divergent melanic pigmentation in benthic and limnetic threespine stickleback.

Pigmentation is an excellent trait to examine patterns of evolutionary change because it is often under natural selection. Benthic and limnetic threespine stickleback (Gasterosteus aculeatus) exhibit distinct pigmentation phenotypes, likely an adaptation to occupation of divergent niches. The genetic architecture of pigmentation in vertebrates appears to be complex. Prior QTL mapping of threespine stickleback pigmentation phenotypes has identified several candidate loci. However-relative to other morphological phenotypes (e.g., spines or lateral plates)-the genetic architecture of threespine stickleback pigmentation remains understudied. Here, we performed QTL mapping for two melanic pigmentation traits (melanophore density and lateral barring) using benthic-limnetic F2 crosses. The two traits mapped to different chromosomes, suggesting a distinct genetic basis. The resulting QTLs were additive, but explained a relatively small fraction of the total variance (~6%). QTLs maps differed by F1 family, suggesting variation in genetic architecture or ability to detect loci of small effect. Functional analysis identified enriched pathways for candidate loci. Several of the resulting candidate loci for pigmentation, including three loci in enriched pathways (bco1, sulf1, and tyms) have been previously indicated to affect pigmentation in other vertebrates. These findings add to a growing body of evidence suggesting pigmentation is often polygenic.

Soft-tissue evidence for homeothermy and crypsis in a Jurassic ichthyosaur.

Ichthyosaurs are extinct marine reptiles that display a notable external similarity to modern toothed whales. Here we show that this resemblance is more than skin deep. We apply a multidisciplinary experimental approach to characterize the cellular and molecular composition of integumental tissues in an exceptionally preserved specimen of the Early Jurassic ichthyosaur Stenopterygius. Our analyses recovered still-flexible remnants of the original scaleless skin, which comprises morphologically distinct epidermal and dermal layers. These are underlain by insulating blubber that would have augmented streamlining, buoyancy and homeothermy. Additionally, we identify endogenous proteinaceous and lipid constituents, together with keratinocytes and branched melanophores that contain eumelanin pigment. Distributional variation of melanophores across the body suggests countershading, possibly enhanced by physiological adjustments of colour to enable photoprotection, concealment and/or thermoregulation. Convergence of ichthyosaurs with extant marine amniotes thus extends to the ultrastructural and molecular levels, reflecting the omnipresent constraints of their shared adaptation to pelagic life.

A complex genetic architecture in zebrafish relatives Danio quagga and D. kyathit underlies development of stripes and spots.

Vertebrate pigmentation is a fundamentally important, multifaceted phenotype. Zebrafish, Danio rerio, has been a valuable model for understanding genetics and development of pigment pattern formation due to its genetic and experimental tractability, advantages that are shared across several Danio species having a striking array of pigment patterns. Here, we use the sister species D. quagga and D. kyathit, with stripes and spots, respectively, to understand how natural genetic variation impacts phenotypes at cellular and organismal levels. We first show that D. quagga and D. kyathit phenotypes resemble those of wild-type D. rerio and several single locus mutants of D. rerio, respectively, in a morphospace defined by pattern variation along dorsoventral and anteroposterior axes. We then identify differences in patterning at the cellular level between D. quagga and D. kyathit by repeated daily imaging during pattern development and quantitative comparisons of adult phenotypes, revealing that patterns are similar initially but diverge ontogenetically. To assess the genetic architecture of these differences, we employ reduced-representation sequencing of second-generation hybrids. Despite the similarity of D. quagga to D. rerio, and D. kyathit to some D. rerio mutants, our analyses reveal a complex genetic basis for differences between D. quagga and D. kyathit, with several quantitative trait loci contributing to variation in overall pattern and cellular phenotypes, epistatic interactions between loci, and abundant segregating variation within species. Our findings provide a window into the evolutionary genetics of pattern-forming mechanisms in Danio and highlight the complexity of differences that can arise even between sister species. Further studies of natural genetic diversity underlying pattern variation in D. quagga and D. kyathit should provide insights complementary to those from zebrafish mutant phenotypes and more distant species comparisons.

Structural and ultrastructural aspects of the skin of large yellow croaker Larimichthys crocea.

The skin color of the large yellow croaker (Larimichthys crocea) is a crucial indicator to determine its economic value. However, the location of pigment cells in the skin structure is uncertain. To determine the pigment cell type in the skin, the vertical order and ultrastructure of pigment cells were examined using light microscopy and transmission electron microscopy. Both dorsal and ventral skins comprise the epidermis, dermis, and hypodermis. Xanthophores, melanophores, and iridophores were observed in the dermis of the dorsal skin, whereas the latter two were in the dermis of the ventral skin. Interestingly, the size of xanthophores in the dorsal skin was significantly smaller than that of xanthophores in the ventral skin; however, the density of dorsal xanthophores was significantly higher than that of ventral xanthophores. The type L-iridophores with large crystalline structures were observed in the uppermost area of the upper pigment layer, which contributed to the strikingly metallic luster shown by the ventral skin. The melanophores were exclusively found in the dorsal skin, offering the purpose of camouflage. Taken together, our results indicated that the pigment cells display different arrangement patterns between dorsal and ventral skin, and the golden color in the ventral skin results from the coexistence of light-reflecting iridophores and light-absorbing xanthophores.

Generation of translucent Xenopus tropicalis through triple knockout of pigmentation genes.

Amphibians generally have three types of pigment cells, namely, melanophores (black and brown), xanthophores (yellow and red), and iridophores (iridescent). Single knockout of the tyr, slc2a7, and hps6 genes in Xenopus tropicalis results in the absence of melanophores, xanthophores, and iridophores, respectively. The generation of triple- knockout (3KO) X. tropicalis for these three genes could allow for observation of internal organs without sacrificing the animals, which would be transparent due to the absence of pigments. In this study, we generated 3KO X. tropicalis, which is one of the most widely used model amphibians, through crossing of a slc2a7 single-knockout frog with a tyr and hps6 double-knockout frog, followed by intercrossing of their offspring. The 3KO tadpoles had transparent bodies like the nop mutant and the frogs had translucent bodies. This translucency allowed us to observe the heart, lungs, stomach, liver, and digestive tract through the ventral body skin without surgery. After intravital staining, 3KO X. tropicalis showed much clearer fluorescent signals of mineralized tissues compared with the wild type. These 3KO X. tropicalis provide a useful mutant line for continuous observation of internal organs and fluorescent signals in the body. In particular, such 3KO frogs would revolutionize fluorescence monitoring in transgenic tadpoles and frogs expressing fluorescent proteins.

Genome mapping of a LYST mutation in corn snakes indicates that vertebrate chromatophore vesicles are lysosome-related organelles.

Reptiles exhibit a spectacular diversity of skin colors and patterns brought about by the interactions among three chromatophore types: black melanophores with melanin-packed melanosomes, red and yellow xanthophores with pteridine- and/or carotenoid-containing vesicles, and iridophores filled with light-reflecting platelets generating structural colors. Whereas the melanosome, the only color-producing endosome in mammals and birds, has been documented as a lysosome-related organelle, the maturation paths of xanthosomes and iridosomes are unknown. Here, we first use 10x Genomics linked-reads and optical mapping to assemble and annotate a nearly chromosome-quality genome of the corn snake Pantherophis guttatus The assembly is 1.71 Gb long, with an N50 of 16.8 Mb and L50 of 24. Second, we perform mapping-by-sequencing analyses and identify a 3.9-Mb genomic interval where the lavender variant resides. The lavender color morph in corn snakes is characterized by gray, rather than red, blotches on a pink, instead of orange, background. Third, our sequencing analyses reveal a single nucleotide polymorphism introducing a premature stop codon in the lysosomal trafficking regulator gene (LYST) that shortens the corresponding protein by 603 amino acids and removes evolutionary-conserved domains. Fourth, we use light and transmission electron microscopy comparative analyses of wild type versus lavender corn snakes and show that the color-producing endosomes of all chromatophores are substantially affected in the LYST mutant. Our work provides evidence characterizing xanthosomes in xanthophores and iridosomes in iridophores as lysosome-related organelles.

Effects of monochromatic lights on the melanophores arrangement in the spotted snakehead fish Channa punctata (Bloch, 1793).

Some freshwater teleost fish have pigment cells whose arrangement and shape are affected by the environment. Natural light has a wide range of light intensity. Fish are sensitive to the background and exposed light colour. Fish body colour is a significant criterion in fixing its market value, whether it is ornamental or edible. By favourable light exposure, a culturist may get a good market value of fish on most ethical grounds. In this study, we recorded the changes in melanophore response with the changes in light colour on Channa punctata. Adult fish were treated with monochromatic lights (darkness, white, blue and red light) for 5 and 28 days. After treatment, their body colour and melanophore size, number, length and the number of dendrites were studied. The results showed a significant influence of monochromatic light on melanophore arrangement in fish skin. The data showed that blue light is appropriate for the overall species colour of photic C. punctata. Continuous black or white light caused severe damage to the fish's appearance.

A new species of the conger eel genus Macrocephenchelys from deep waters of the Arabian Sea.

A new species of the genus Macrocephenchelys is described herein based on a single specimen collected from the deep-sea trawl landing at Kalamukku fish landing centre, Kerala coast, Arabian Sea. The new species is distinguished by having a dorsal-fin origin behind the middle of pectoral fin, a larger head, shorter trunk, larger gill opening, dorsal surface of body with dark-brown colour and ventral surface of head and belly with numerous patches of melanophores before anus, vertebrae 14-30-151. The new species shares most of the characteristics with Macrocephenchelys brevirostris but differs from it by having a more anterior dorsal-fin origin (vs. over the tip or slightly behind the pectoral-fin tip), larger head [15.3% total length (TL) vs. 10.5%-13.9% TL, 53.2% pre-anal length (PAL) vs. 35.8%-47.6% PAL], shorter trunk length (13.6% TL vs. 14.4%-20.6% TL, 47.3% PAL vs. 52.4%-66.2% PAL); further it shows 7.9%-8.1% genetic divergence from the sequences of M. brevirostris.

Exploring the mechanisms and impacts of melatonin on fish colouration.

The pineal hormone melatonin is a multi-functional molecule with a recognized role in pigment aggregation in chromatophores, mediating its actions through binding to subtypes of its specific receptors. Since its discovery, melatonin has been known to be responsible for pigment aggregation towards the cell centre in fishes, including their embryos, as an adaptation to reduced light and thus results in pale body colouration. Diversity exists in the sensitivity of melanophores towards melatonin at interspecies, intraspecific levels, seasons, and amongst chromatophores at different regions of the animal body. In most of the fishes, melatonin leads to their skin paling at night. It is indicated that the melatonin receptors have characteristically maintained to show the same aggregating effects in fishes and other vertebrates in the evolutionary hierarchy. However, besides this aggregatory effect, melatonin is also responsible for pigment dispersion in certain fishes. Here is the demand in our review to explore further the nature of the dispersive behaviour of melatonin through the so-called ß-melatonin receptors. It is clear that the pigment translocations in lower vertebrates under the effect of melatonin are mediated through the melatonin receptors coupled with other hormonal receptors as well. Therefore, being richly supplied with a variety of receptors, chromatophores and melanocytes can be used as in vitro test models for pharmacological applications of known and novel drugs. In this review, we present diverse effects of melatonin on chromatophores of fishes in particular with appropriate implications on most of the recent findings.

Immunoglobulin superfamily receptor Junctional adhesion molecule 3 (Jam3) requirement for melanophore survival and patterning during formation of zebrafish stripes.

Adhesive interactions are essential for tissue patterning and morphogenesis yet difficult to study owing to functional redundancies across genes and gene families. A useful system in which to dissect roles for cell adhesion and adhesion-dependent signaling is the pattern formed by pigment cells in skin of adult zebrafish, in which stripes represent the arrangement of neural crest derived melanophores, cells homologous to melanocytes. In a forward genetic screen for adult pattern defects, we isolated the pissarro (psr) mutant, having a variegated phenotype of spots, as well as defects in adult fin and lens. We show that psr corresponds to junctional adhesion protein 3b (jam3b) encoding a zebrafish orthologue of the two immunoglobulin-like domain receptor JAM3 (JAM-C), known for roles in adhesion and signaling in other developing tissues, and for promoting metastatic behavior of human and murine melanoma cells. We found that zebrafish jam3b is expressed post-embryonically in a variety of cells including melanophores, and that jam3b mutants have defects in melanophore survival. Jam3b supported aggregation of cells in vitro and was required autonomously by melanophores for an adherent phenotype in vivo. Genetic analyses further indicated both overlapping and non-overlapping functions with the related receptor, Immunoglobulin superfamily 11 (Igsf11) and Kit receptor tyrosine kinase. These findings suggest a model for Jam3b function in zebrafish melanophores and hint at the complexity of adhesive interactions underlying pattern formation.

The minimal gap-junction network among melanophores and xanthophores required for stripe pattern formation in zebrafish.

Connexin 39.4 (Cx39.4) and connexin 41.8 (Cx41.8), two gap-junction proteins expressed in both melanophores and xanthophores, are crucial for the intercellular communication among pigment cells that is necessary for generating the stripe pigment pattern of zebrafish. We have previously characterized the gap-junction properties of Cx39.4 and Cx41.8, but how these proteins contribute to stripe formation remains unclear; this is because distinct types of connexins potentially form heteromeric gap junctions, which precludes accurate elucidation of individual connexin functions in vivo Here, by arranging Cx39.4 and Cx41.8 expression in pigment cells, we have identified the simplest gap-junction network required for stripe generation: Cx39.4 expression in melanophores is required but expression in xanthophores is not necessary for stripe patterning, whereas Cx41.8 expression in xanthophores is sufficient for the patterning, and Cx41.8 expression in melanophores might stabilize the stripes. Moreover, patch-clamp recordings revealed that Cx39.4 gap junctions exhibit spermidine-dependent rectification property. Our results suggest that Cx39.4 facilitates the crucial cell-cell interactions between melanophores and xanthophores that mediate a unidirectional activation-signal transfer from xanthophores to melanophores, which is essential for melanophore survival.

CRISPR Knockouts of pmela and pmelb Engineered a Golden Tilapia by Regulating Relative Pigment Cell Abundance.

Premelanosome protein (pmel) is a key gene for melanogenesis. Mutations in this gene are responsible for white plumage in chicken, but its role in pigmentation of fish remains to be demonstrated. In this study, we found that most fishes have 2 pmel genes arising from the teleost-specific whole-genome duplication. Both pmela and pmelb were expressed at high levels in the eyes and skin of Nile tilapia. We mutated both genes in tilapia using CRISPR/Cas9. Homozygous mutation of pmela resulted in yellowish body color with weak vertical bars and a hypopigmented retinal pigment epithelium (RPE) due to significantly reduced number and size of melanophores. In contrast, we observed an increased number and size of xanthophores in mutants compared to wild-type fish. Homozygous mutation of pmelb resulted in a similar, but milder phenotype than pmela-/- mutants. Double mutation of pmela and pmelb resulted in loss of additional melanophores compared to the pmela-/- mutants, and also an increase in the number and size of xanthophores, producing a golden body color. The RPE pigmentation of pmela-/-;pmelb-/- was similar to pmela-/- mutants, with much less pigmentation than pmelb-/- mutants and wild-type fish. Taken together, our results indicate that, although both pmel genes are important for the formation of body color in tilapia, pmela plays a more important role than pmelb. To our knowledge, this is the first report on mutation of pmelb or both pmela;pmelb in fish. Studies on these mutants suggest new strategies for breeding golden tilapia, and also provide a new model for studies of pmel function in vertebrates.

Fate plasticity and reprogramming in genetically distinct populations of Danio leucophores.

Understanding genetic and cellular bases of adult form remains a fundamental goal at the intersection of developmental and evolutionary biology. The skin pigment cells of vertebrates, derived from embryonic neural crest, are a useful system for elucidating mechanisms of fate specification, pattern formation, and how particular phenotypes impact organismal behavior and ecology. In a survey of Danio fishes, including the zebrafish Danio rerio, we identified two populations of white pigment cells-leucophores-one of which arises by transdifferentiation of adult melanophores and another of which develops from a yellow-orange xanthophore or xanthophore-like progenitor. Single-cell transcriptomic, mutational, chemical, and ultrastructural analyses of zebrafish leucophores revealed cell-type-specific chemical compositions, organelle configurations, and genetic requirements. At the organismal level, we identified distinct physiological responses of leucophores during environmental background matching, and we showed that leucophore complement influences behavior. Together, our studies reveal independently arisen pigment cell types and mechanisms of fate acquisition in zebrafish and illustrate how concerted analyses across hierarchical levels can provide insights into phenotypes and their evolution.

Endothelin receptor Aa regulates proliferation and differentiation of Erb-dependent pigment progenitors in zebrafish.

Skin pigment patterns are important, being under strong selection for multiple roles including camouflage and UV protection. Pigment cells underlying these patterns form from adult pigment stem cells (APSCs). In zebrafish, APSCs derive from embryonic neural crest cells, but sit dormant until activated to produce pigment cells during metamorphosis. The APSCs are set-aside in an ErbB signaling dependent manner, but the mechanism maintaining quiescence until metamorphosis remains unknown. Mutants for a pigment pattern gene, parade, exhibit ectopic pigment cells localised to the ventral trunk, but also supernumerary cells restricted to the Ventral Stripe. Contrary to expectations, these melanocytes and iridophores are discrete cells, but closely apposed. We show that parade encodes Endothelin receptor Aa, expressed in the blood vessels, most prominently in the medial blood vessels, consistent with the ventral trunk phenotype. We provide evidence that neuronal fates are not affected in parade mutants, arguing against transdifferentiation of sympathetic neurons to pigment cells. We show that inhibition of BMP signaling prevents specification of sympathetic neurons, indicating conservation of this molecular mechanism with chick and mouse. However, inhibition of sympathetic neuron differentiation does not enhance the parade phenotype. Instead, we pinpoint ventral trunk-restricted proliferation of neural crest cells as an early feature of the parade phenotype. Importantly, using a chemical genetic screen for rescue of the ectopic pigment cell phenotype of parade mutants (whilst leaving the embryonic pattern untouched), we identify ErbB inhibitors as a key hit. The time-window of sensitivity to these inhibitors mirrors precisely the window defined previously as crucial for the setting aside of APSCs in the embryo, strongly implicating adult pigment stem cells as the source of the ectopic pigment cells. We propose that a novel population of APSCs exists in association with medial blood vessels, and that their quiescence is dependent upon Endothelin-dependent factors expressed by the blood vessels.

Analysis of pigment cell composition, pigment content, tyrosinase content and activity of three kinds of loaches Misgurnus anguillicaudatus from Poyang Lake.

Pigment cell composition, pigment content, tyrosinase content and activity analysis were investigated on three kinds of loaches Misgurnus anguillicaudatus: big blackspot loaches (BBL), small blackspot loaches (SBL) and non-blackspot loaches (NBL), from Poyang Lake. Results showed that there were three types of skin pigment cells, namely melanophores, xanthophores and iridophores. Melanophores in dorsum were more than those in abdomen. Melanophore cytosomes in BBL were larger than those in SBL and NBL, and melanosomes were the largest in stage four. The melanophores in dorsal skin of SBL or NBL were small cell bodies, spindle-like and in chain distribution. There was an extremely significant difference in melanin content in BBL between the dorsum and abdomen (P < 0.01). There were no significant differences in melanin abdominal content, lutein and carotenoid contents among three kinds of loaches (P > 0.05). In dorsal skin, tyrosinase content was the highest in BBL, and it was significantly lower in NBL than in BBL and SBL (P < 0.01). This study reveals the differences in pigment and tyrosinase content in three kinds of loaches and provides a theoretical basis for further study of the mechanism of black spot formation.

Generation of no-yellow-pigment Xenopus tropicalis by slc2a7 gene knockout.

BACKGROUND: Amphibians possess three kinds of dermal chromatophore: melanophores, iridophores, and xanthophores. Knockout Xenopus tropicalis that lack the pigmentation of melanophores and iridophores have been reported. The identification of the causal genes for xanthophore pigmentation or differentiation could lead to the creation of a see-through frog without three chromatophores. The genes causing xanthophore differentiation mutants are slc2a11b and slc2a15b in Japanese medaka (Oryzias latipes). RESULTS: To obtain a heritable line of X tropicalis mutants without yellow pigment, we generated slc2a7 and slc2a15a knockout animals because they have the greatest similarity to the O latipes slc2a11b and slc2a15b genes. The slc2a7 knockout frog had a bluish skin and there were no visible yellow pigments in stereo microscope and skin section observations. Furthermore, no pterinosomes, which are characteristic of xanthophores, were observed via transmission electron microscopy in the skin of knockout animals. CONCLUSIONS: We report the successful generation of a heritable no-yellow-pigment X tropicalis mutant after knock out of the slc2a7 gene. This finding will enable the creation of a see-through frog with no chromatophores.

Method for disarranging the pigment pattern of zebrafish by optogenetics.

To investigate the spatiotemporal dynamics of skin pattern formation, we developed a simple method for artificially disarranging the placement of all three pigment cell types in the body trunk of zebrafish (Danio rerio). We generated transgenic fish with melanophores that ectopically expressed a variant of channelrhodopsin-2 (ChR2). Blue light (BL) irradiation induced melanophore depolarization and random migration; the latter resulted in the disarrangement of the two other pigment cell types (xanthophores and iridophores). This BL disarrangement (BLD) method was effective in both young and adult fish, but it did not affect the initial placement of pigment cells in juvenile fish (approximately 5 weeks post-fertilization). Irradiation with BL was not harmful to cells, and the patterning process immediately resumed when BL was switched off. Using the BLD method, we demonstrated that interactions between pigment cells determined stripe width in the absence of any pre-set positional cues, while the initial horizontal alignment of iridophores determined their directionality. The BLD method can be adapted to any zebrafish skin-pattern mutant, providing a novel tool for analyzing pattern formation mechanisms under a variety of conditions and facilitating further study in this field.