An outstanding problem in the study of vertebrate development is the identification of the genes that direct neural crest precursor cells to adopt and maintain specific differentiated cell fates. In an effort to identify such genes, we have carried out a mutagenesis screen in zebrafish and isolated mutants that lack neural crest-derived melanophores. In this manuscript we describe the phenotype of one such mutant, touchtone(b722) (tct), and the map position of the gene it defines. Analysis of expression of dopachrome tautomerase (dct) and microphthalmia (mitfa) suggests that melanophore precursors are specified normally in homozygous tct mutants. However, differentiated melanophores are pale, small, and about half of them have disappeared by 48 h of development, apparently by cell death. We show that melanophores require Tct function cell autonomously. Signals from the receptor tyrosine kinase receptor C-kit are essential for survival of melanophores in zebrafish and mammals. However, differences in the phenotypes of tct and c-kit homozygous mutants, and an absence of interaction between c-kit and tct heterozygotes, suggest that Tct functions independently of the C-kit pathway. Other neural crest derivatives, including other pigment cell types, appear normal in tct mutants. Interestingly, tct mutant embryos undergo a temporary period of near complete paralyzis during the second day of development, although markers of axons of motor and sensory neurons look normal in this period. A fraction of tct(b722) mutants survive to adulthood, but mutant adults are small, indicating a role for Tct in post-larval growth. The tct gene maps to a small interval near a telomere of chromosome 18. Thus, we have identified a zebrafish gene that when mutated produces semi-viable offspring and that may serve as a model of human diseases that have both pigmentation and neurological symptoms.

The organization of the cytoplasm is regulated by molecular motors, which transport organelles and other cargoes along cytoskeleton tracks. In this work, we use single particle tracking to study the in vivo regulation of the transport driven by myosin-V along actin filaments in Xenopus laevis melanophores. Melanophores have pigment organelles or melanosomes, which, in response to hormones, disperse in the cytoplasm or aggregate in the perinuclear region. We followed the motion of melanosomes in cells treated to depolymerize microtubules during aggregation and dispersion, focusing the analysis on the dynamics of these organelles in a time window not explored before to our knowledge. These data could not be explained by previous models that only consider active transport. We proposed a transport-diffusion model in which melanosomes may detach from actin tracks and reattach to nearby filaments to resume the active motion after a given time of diffusion. This model predicts that organelles spend approximately 70% and 10% of the total time in active transport during dispersion and aggregation, respectively. Our results suggest that the transport along actin filaments and the switching from actin to microtubule networks are regulated by changes in the diffusion time between periods of active motion driven by myosin-V.

A model of the gene-regulatory-network (GRN), governing growth, survival, and differentiation of melanocytes, has emerged from studies of mouse coat color mutants and melanoma cell lines. In this model, Transcription Factor Activator Protein 2 alpha (TFAP2A) contributes to melanocyte development by activating expression of the gene encoding the receptor tyrosine kinase Kit. Next, ligand-bound Kit stimulates a pathway activating transcription factor Microphthalmia (Mitf), which promotes differentiation and survival of melanocytes by activating expression of Tyrosinase family members, Bcl2, and other genes. The model predicts that in both Tfap2a and Kit null mutants there will be a phenotype of reduced melanocytes and that, because Tfap2a acts upstream of Kit, this phenotype will be more severe, or at least as severe as, in Tfap2a null mutants in comparison to Kit null mutants. Unexpectedly, this is not the case in zebrafish or mouse. Because many Tfap2 family members have identical DNA-binding specificity, we reasoned that another Tfap2 family member may work redundantly with Tfap2a in promoting Kit expression. We report that tfap2e is expressed in melanoblasts and melanophores in zebrafish embryos and that its orthologue, TFAP2E, is expressed in human melanocytes. We provide evidence that Tfap2e functions redundantly with Tfap2a to maintain kita expression in zebrafish embryonic melanophores. Further, we show that, in contrast to in kita mutants where embryonic melanophores appear to differentiate normally, in tfap2a/e doubly-deficient embryonic melanophores are small and under-melanized, although they retain expression of mitfa. Interestingly, forcing expression of mitfa in tfap2a/e doubly-deficient embryos partially restores melanophore differentiation. These findings reveal that Tfap2 activity, mediated redundantly by Tfap2a and Tfap2e, promotes melanophore differentiation in parallel with Mitf by an effector other than Kit. This work illustrates how analysis of single-gene mutants may fail to identify steps in a GRN that are affected by the redundant activity of related proteins.

1. GR128107 (3-(1-acetyl-3-methyl-piperidine)-5-methoxyindole) has previously been reported to be a competitive melatonin receptor antagonist in blocking melatonin inhibition of [3H]-dopamine release from rabbit retina, a response mediated by the MT2 receptor subtype. 2. GR128107, like melatonin, induced a rapid (maximum response in 60-90 min) pigment aggregation in a clonal line of Xenopus laevis melanophores. GR128107 behaved as a partial agonist (pEC50 8.58+/-0.03, n=3) with an Emax of 0.83 (relative to melatonin, pEC50 10.09+/-0.03, n=3). 3. The concentration-response curve for pigment granule aggregation to both melatonin and GR128107 was displaced in a parallel, rightward manner by melatonin receptor antagonists with very similar potencies; estimated pKB RJ252 (against melatonin 4.60/against GR128107 4.54) < GR135533 (6.40/6.14) < Luzindole (6.45/6.49) < S20929 (6.58/6.65) < 4-P-PDOT (6.73/6.85). 4. Both melatonin- and GR128107-induced pigment granule aggregation was prevented by pretreatment of melanophores with pertussis toxin (10-1000 ng ml(-1)). 5. Prolonged pre-treatment of melanophores with melatonin desensitized the pigment aggregation response to GR128107. In desensitized cells, the maximal aggregation produced by GR128107 was only 0.27+/-0.01 (n=4) and the pEC50 was reduced (vehicle 8.57+/-0.12; melatonin pre-treated 7.84+/-0.09, n=4). The maximal response to melatonin in desensitized melanophores was unchanged but the pEC50 was reduced (vehicle 10.49+/-0.03; melatonin pre-treated 9.83+/-0.04, n=4). 6. These results demonstrate that GR128107 induces pigment granule aggregation in Xenopus melanophores by activating a cell membrane melatonin receptor coupled via a pertussis toxin-sensitive G-protein. 7. The partial agonist activity of GR128107 in melanophores may be apparent because of the very high density of melatonin receptors in these cells (Bmax 1223 fmol mg protein(-1)) compared to the low density of sites in rabbit retina (Bmax 3.1 fmol mg protein(-1)). This suggestion is supported by the finding that GR128107, like melatonin, acted as a full agonist and inhibited forskolin-stimulation of cyclic AMP accumulation in NIH-3T3 cells expressing a high density of human mt1 or MT2 receptors.

Berberine is an active compound of Berberis vulgaris (Daruhaldi) with known multiple pharmacological activities, including antimicrobial, antiviral, anti-inflammatory, cholesterol-lowering, and anticancer effects. The present work aimed to study the ultrastructural effects of berberine to determine its skin-darkening potential using Bufo melanostictus melanophores, which has not been done to date. Light and electron microscopic analysis of isolated dorsal skin melanophores of B. melanostictus has been done after treatment with various concentrations of berberine, along with specific antagonists and agonists of β-adrenoceptors in order to explore the mechanism of action of berberine-induced skin darkening. The results showed that the number of melanophores with melanin-loaded dendrites increased in the subepidermal layer significantly in berberine-treated skin pieces in a dose-dependent manner leading to skin darkening. Highly electron-dense melanosomes of Stage IV increased considerably due to the enhanced process of melanization. These effects were found to be antagonized by propranolol, and were also found to be highly potentiated by isoprenaline, which is a specific β-adrenoceptor agonist. The findings show that berberine possesses a skin-darkening potential and could be used as a safe melanogenic agent for the treatment of hypopigmentation disorders or vitiligo.

α-MSH and light exert a dispersing effect on pigment granules of Xenopus laevis melanophores; however, the intracellular signaling pathways are different. Melatonin, a hormone that functions as an internal signal of darkness for the organism, has opposite effects, aggregating the melanin granules. Because light functions as an important synchronizing signal for circadian rhythms, we further investigated the effects of both hormones on genes related to the circadian system, namely, Per1 (one of the clock genes) and the melanopsins, Opn4x and Opn4m (photopigments). Per1 showed temporal oscillations, regardless of the presence of melatonin or α-MSH, which slightly inhibited its expression. Melatonin effects on melanopsins depend on the time of application: if applied in the photophase it dramatically decreased Opn4x and Opn4m expressions, and abolished their temporal oscillations, opposite to α-MSH, which increased the melanopsins' expressions. Our results demonstrate that unlike what has been reported for other peripheral clocks and cultured cells, medium changes or hormones do not play a major role in synchronizing the Xenopus melanophore population. This difference is probably due to the fact that X. laevis melanophores possess functional photopigments (melanopsins) that enable these cells to primarily respond to light, which triggers melanin dispersion and modulates gene expression.

Melanophores move pigment organelles (melanosomes) from the cell center to the periphery and vice-versa. These bidirectional movements require cytoplasmic microtubules and microfilaments and depend on the function of microtubule motors and a myosin. Earlier we found that melanosomes purified from Xenopus melanophores contain the plus end microtubule motor kinesin II, indicating that it may be involved in dispersion (Rogers, S.L., I.S. Tint, P.C. Fanapour, and V.I. Gelfand. 1997. Proc. Natl. Acad. Sci. USA. 94: 3720-3725). Here, we generated a dominant-negative construct encoding green fluorescent protein fused to the stalk-tail region of Xenopus kinesin-like protein 3 (Xklp3), the 95-kD motor subunit of Xenopus kinesin II, and introduced it into melanophores. Overexpression of the fusion protein inhibited pigment dispersion but had no effect on aggregation. To control for the specificity of this effect, we studied the kinesin-dependent movement of lysosomes. Neither dispersion of lysosomes in acidic conditions nor their clustering under alkaline conditions was affected by the mutant Xklp3. Furthermore, microinjection of melanophores with SUK4, a function-blocking kinesin antibody, inhibited dispersion of lysosomes but had no effect on melanosome transport. We conclude that melanosome dispersion is powered by kinesin II and not by conventional kinesin. This paper demonstrates that kinesin II moves membrane-bound organelles.

Vertebrates have a central clock and also several peripheral clocks. Light responses might result from the integration of light signals by these clocks. The dermal melanophores of Xenopus laevis have a photoreceptor molecule denominated melanopsin (OPN4x). The mechanisms of the circadian clock involve positive and negative feedback. We hypothesize that these dermal melanophores also present peripheral clock characteristics. Using quantitative PCR, we analyzed the pattern of temporal expression of Opn4x and the clock genes Per1, Per2, Bmal1, and Clock in these cells, subjected to a 14-h light:10-h dark (14L:10D) regime or constant darkness (DD). Also, in view of the physiological role of melatonin in the dermal melanophores of X. laevis, we determined whether melatonin modulates the expression of these clock genes. These genes show a time-dependent expression pattern when these cells are exposed to 14L:10D, which differs from the pattern observed under DD. Cells kept in DD for 5 days exhibited overall increased mRNA expression for Opn4x and Clock, and a lower expression for Per1, Per2, and Bmal1. When the cells were kept in DD for 5 days and treated with melatonin for 1 h, 24 h before extraction, the mRNA levels tended to decrease for Opn4x and Clock, did not change for Bmal1, and increased for Per1 and Per2 at different Zeitgeber times (ZT). Although these data are limited to one-day data collection, and therefore preliminary, we suggest that the dermal melanophores of X. laevis might have some characteristics of a peripheral clock, and that melatonin modulates, to a certain extent, melanopsin and clock gene expression.

The genes that control development of embryonic melanocytes are poorly defined. Although transcription factor Ap-2alpha is expressed in neural crest (NC) cells, its role in development of embryonic melanocytes and other neural crest derivatives is unclear because mouse Ap-2alpha mutants die before melanogenesis. We show that zebrafish embryos injected with morpholino antisense oligonucleotides complementary to ap-2alpha (ap-2alpha MO) complete early morphogenesis normally and have neural crest cells. Expression of c-kit, which encodes the receptor for the Steel ligand, is reduced in these embryos, and, similar to zebrafish c-kit mutant embryos, embryonic melanophores are reduced in number and migration. The effects of ap-2alpha MO injected into heterozygous and homozygous c-kit mutants support the notion that Ap-2alpha works through C-kit and additional target genes to mediate melanophore cell number and migration. In contrast to c-kit mutant embryos, in ap-2alpha MO-injected embryos, melanophores are small and under-pigmented, and unexpectedly, analysis of mosaic embryos suggests Ap-2alpha regulates melanophore differentiation through cell non-autonomous targets. In addition to melanophore phenotypes, we document reduction of other neural crest derivatives in ap-2alpha MO-injected embryos, including jaw cartilage, enteric neurons, and sympathetic neurons. These results reveal that Ap-2alpha regulates multiple steps of melanophore development, and is required for development of other neuronal and non-neuronal neural crest derivatives.

Skin pigment patterns of vertebrates are a classic system for understanding fundamental mechanisms of morphogenesis, differentiation, and pattern formation, and recent studies of zebrafish have started to elucidate the cellular interactions and molecular mechanisms underlying these processes. In this species, horizontal dark stripes of melanophores alternate with light interstripes of yellow or orange xanthophores and iridescent iridophores. We showed previously that the highly conserved zinc finger protein Basonuclin-2 (Bnc2) is required in the environment in which pigment cells reside to promote the development and maintenance of all three classes of pigment cells; bnc2 mutants lack body stripes and interstripes. Previous studies also revealed that interactions between melanophores and xanthophores are necessary for organizing stripes and interstripes. Here we show that bnc2 promotes melanophore and xanthophore development by regulating expression of the growth factors Kit ligand a (Kitlga) and Colony stimulating factor-1 (Csf1), respectively. Yet, we found that rescue of melanophores and xanthophores was insufficient for the recovery of stripes in the bnc2 mutant. We therefore asked whether bnc2-dependent iridophores might contribute to stripe and interstripe patterning as well. We found that iridophores themselves express Csf1, and by ablating iridophores in wild-type and mutant backgrounds, we showed that iridophores contribute to organizing both melanophores and xanthophores during the development of stripes and interstripes. Our results reveal an important role for the cellular environment in promoting adult pigment pattern formation and identify new components of a pigment-cell autonomous pattern-generating system likely to have broad implications for understanding how pigment patterns develop and evolve.

We used melanophores, cells specialized for regulated organelle transport, to study signaling pathways involved in the regulation of transport. We transfected immortalized Xenopus melanophores with plasmids encoding epitope-tagged inhibitors of protein phosphatases and protein kinases or control plasmids encoding inactive analogues of these inhibitors. Expression of a recombinant inhibitor of protein kinase A (PKA) results in spontaneous pigment aggregation. alpha-Melanocyte-stimulating hormone (MSH), a stimulus which increases intracellular cAMP, cannot disperse pigment in these cells. However, melanosomes in these cells can be partially dispersed by PMA, an activator of protein kinase C (PKC). When a recombinant inhibitor of PKC is expressed in melanophores, PMA-induced pigment dispersion is inhibited, but not dispersion induced by MSH. We conclude that PKA and PKC activate two different pathways for melanosome dispersion. When melanophores express the small t antigen of SV-40 virus, a specific inhibitor of protein phosphatase 2A (PP2A), aggregation is completely prevented. Conversely, overexpression of PP2A inhibits pigment dispersion by MSH. Inhibitors of protein phosphatase 1 and protein phosphatase 2B (PP2B) do not affect pigment movement. Therefore, melanosome aggregation is mediated by PP2A.

The present study was aimed to evaluate the effect of municipal wastewater of Tung Dhab drain on morphology of scale melanophores in freshwater fish Labeo rohita (Ham.). Chronic, non-renewal toxicity tests were performed with sub lethal concentrations (17.7, 26.6 and 35.4%) of wastewater for exposure durations of 15, 30 and 60 days. Recovery experiments were also performed for duration of 60 days. The scales were removed, processed and diameters of melanophores were measured using an ocular micrometer. The results showed concentration and duration dependent changes in melanophore morphology (size and dendricity) in experimental fish as compared to control fish. The values of mean melanophore size index (MMSI) varied from 5.37 ± 0.49 (17.7%) to 12.12 ± 0.81 (35.5%) in comparison to control values of 4.32 ± 0.32 and 4.55 ± 0.29 for 15 and 60 days respectively. The recovery experiments suggested that observed dark colouration due to pigment translocation is reversible, even after chronic exposure for 60 days.

"White pigment cells" are derived from melanophore precursors and contain both melanophore-specific and iridophore-specific pigment organelles. Whereas melanophores differentiate in the wild type regenerating tail, white pigment cells appear in the regenerating tail in the periodic albino mutant (a(p)/a(p)) of Xenopus laevis. The localization and density of white pigment cells in the mutant regenerating tail are similar to those of melanophores in the wild type regenerating tail. Here, white pigment cells in the mutant regenerating tail have been compared with melanophores in the wild type regenerating tail in the presence of phenylthiourea (PTU), which inhibits melanosome maturation in melanophores but does not affect reflecting platelet formation in white pigment cells. Ultrastructural analysis shows that reflecting platelet formation in white pigment cells is different from that in iridophores. Reflecting platelets in iridophores are formed from spherical vesicles with electron-dense material, whereas they are formed from stage II melanosomes characteristic of melanophore precursors in white pigment cells. Ultrastructural features of pigment organelles, except reflecting platelets, are similar between mutant melanophores and white pigment cells. In an attempt to identify specific genes in white pigment cells, a subtracted cDNA library enriched for mutant cDNAs has been prepared. Subtracted cDNA fragments have been cloned and selected by whole mount in situ hybridization. Among cDNA fragments examined so far, the ferritin H subunit gene is specifically expressed in white pigment cells, but not in melanophores. Pigment organellogenesis and specific gene expression in white pigment cells are also discussed.

Pituitary adenylate cyclase-activating polypeptide (PACAP) administered to tilapia melanophores ex-vivo causes significant pigment aggregation and this is a newly identified function for this peptide in fish. The G-protein coupled receptors (GPCRs), adcyap1r1a (encoding Pac1a) and vipr2a (encoding Vpac2a), are the only receptors in melanophores with appreciable levels of expression and are significantly (p < 0.05) down-regulated in the absence of light. Vpac2a is activated exclusively by peptide histidine isoleucine (PHI), which suggests that Pac1a mediates the melanin aggregating effect of PACAP on melanophores. Paradoxically activation of Pac1a with PACAP caused a rise in cAMP, which in fish melanophores is associated with melanin dispersion. We hypothesise that the duplicate adcyap1ra and vipr2a genes in teleosts have acquired a specific role in skin and that the melanin aggregating effect of PACAP results from the interaction of Pac1a with Ramp that attenuates cAMP-dependent PKA activity and favours the Ca(2+)/Calmodulin dependent pathway.

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.

Pristella maxillaris is known as the X-ray fish based on its translucent body. However, the morphological characteristics and the molecular regulatory mechanisms of these translucent bodies are still unknown. In this study, the following three phenotypes, a black-and-gray body color or wild-type (WT), a silvery-white body color defined as mutant I (MU1), and a fully transparent body with a visible visceral mass named as mutant II (MU2), were investigated to analyze their chromatophores and molecular mechanisms. The variety and distribution of pigment cells in the three phenotypes of P. maxillaris significantly differed by histological assessment. Three types of chromatophores (melanophores, iridophores, and xanthophores) were observed in the WT, whereas MU1 fish were deficient in melanophores, and MU2 fish lacked melanophores and iridophores. Transcriptome sequencing of the skin and peritoneal tissues of P. maxillaris identified a total of 166,089 unigenes. After comparing intergroup gene expression levels, more than 3,000 unigenes with significantly differential expression levels were identified among three strains. Functional annotation and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of the differentially expressed genes (DEGs) identified a number of candidates melanophores and iridophores genes that influence body color. Some DEGs that were identified using transcriptome analysis were confirmed by quantitative real-time PCR. This study serves as a global survey of the morphological characteristics and molecular mechanism of different body colors observed in P. maxillaris and thus provides a valuable theoretical foundation for the molecular regulation of the transparent phenotype.

Melanin granule (melanosome) dispersion within Xenopus laevis melanophores is evoked either by light or alpha-MSH. We have previously demonstrated that the initial biochemical steps of light and alpha-MSH signaling are distinct, since the increase in cAMP observed in response to alpha-MSH was not seen after light exposure. cAMP concentrations in response to alpha-MSH were significantly lower in cells pre-exposed to light as compared to the levels in dark-adapted melanophores. Here we demonstrate the presence of an adenylyl cyclase (AC) in the Xenopus melanophore, similar to the mammalian type IX which is inhibited by Ca(2+)-calmodulin-activated phosphatase. This finding supports the hypothesis that the cyclase could be negatively modulated by a light-promoted Ca(2+) increase. In fact, the activity of calcineurin PP2B phosphatase was increased by light, which could result in AC IX inhibition, thus decreasing the response to alpha-MSH. St-Ht31, a disrupting agent of protein kinase A (PKA)-anchoring kinase A protein (AKAP) complex totally blocked the melanosome dispersing response to alpha-MSH, but did not impair the photo-response in Xenopus melanophores. Sequence comparison of a melanophore AKAP partial clone with GenBank sequences showed that the anchoring protein was a gravin-like adaptor previously sequenced from Xenopus non-pigmentary tissues. Co-immunoprecipitation of Xenopus AKAP and the catalytic subunit of PKA demonstrated that PKA is associated with AKAP and it is released in the presence of alpha-MSH. We conclude that in X. laevis melanophores, AKAP12 (gravin-like) contains a site for binding the inactive PKA thus compartmentalizing PKA signaling and also possesses binding sites for PKC. Light diminishes alpha-MSH-induced increase of cAMP by increasing calcineurin (PP2B) activity, which in turn inhibits adenylyl cyclase type IX, and/or by activating PKC, which phosphorylates the gravin-like molecule, thus destabilizing its binding to the cell membrane.

The periodic albino mutant of Xenopus laevis is a recessive mutant, in which reduced amounts of melanin appear in the retinal pigment epithelium (RPE) and in melanophores at the late embryonic stage, after which both RPE and melanophores gradually depigment. Three types of pigment cells (melanophores, iridophores and xanthophores) have been reported to be affected in this albino. However, the causative gene of the periodic albinism remains unknown. Hermansky-Pudlak syndrome (HPS) is an autosomal recessive disorder that affects humans and mice, which is caused by defective biogenesis of lysosome-related organelles (LROs). Two subgenomes (L and S) are present in the allotetraploid frog X. laevis. Comparison of genes between the chromosomes 1L and 1S revealed that the HPS type 4 (hps4) gene was present only in chromosome 1L. In the albino mutant, a 1.9 kb genomic deletion in the hps4.L gene including exons 7 and 8 caused a premature stop codon to create a truncated Hps4 protein. Injection of wild-type hps4.L mRNA into mutant embryos rescued the albino phenotype. These findings indicate that hps4 is a causative gene for the periodic albinism in X. laevis. The phenotype of this mutant should be reassessed from the perspective of LRO biogenesis.

Pigment cells of the zebrafish, Danio rerio, offer an exceptionally tractable system for studying the genetic and cellular bases of cell fate decisions. In the zebrafish, neural crest cells generate three types of pigment cells during embryogenesis: yellow xanthophores, iridescent iridophores and black melanophores. In this study, we present evidence for a model whereby melanophores and iridophores descend from a common precursor whose fate is regulated by an interplay between the transcription factors Mitf and Foxd3. Loss of mitfa, a key regulator of melanophore development, resulted in supernumerary ectopic iridophores while loss of foxd3, a mitfa repressor, resulted in fewer iridophores. Double mutants showed a restoration of iridophores, suggesting that one of Foxd3's roles is to suppress mitfa to promote iridophore development. Foxd3 co-localized with pnp4a, a novel marker of early iridophore development, and was necessary for its expression. A considerable overlap was found between iridoblast and melanoblast markers but not xanthoblast markers, which resolved as cells began to differentiate. Cell lineage analyses using the photoconvertible marker, EosFP, revealed that both melanophores and iridophores develop from a mitfa+ precursor. Taken together, our data reveal a Foxd3/mitfa transcriptional switch that governs whether a bi-potent pigment precursor will attain either an iridophore or a melanophore fate.

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.