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. 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 transcription factor Sox10 is required for the specification, migration and survival of all nonectomesenchymal neural crest derivatives including melanophores. sox10-/- zebrafish lack expression of the transcription factor mitfa, which itself is required for melanophore development. We demonstrate that the zebrafish mitfa promoter has sox10 binding sites necessary for activity in vitro, consistent with studies using mammalian cell cultures that have shown that Sox10 directly regulates Mitf expression. In addition, we demonstrate that these sites are necessary for promoter activity in vivo. We show that reintroduction of mitfa expression in neural crest cells can rescue melanophore development in sox10-/- embryos. This rescue of melanophores in sox10-/- embryos is quantitatively indistinguishable from rescue in mitfa-/- embryos. These findings show that the essential function of sox10 in melanophore development is limited to transcriptional regulation of mitfa. We propose that the dominant melanophore phenotype in Waardenburg syndrome IV individuals with SOX10 mutations is likely to result from failure to activate MITF in the normal number of melanoblasts.

Mutations in the zebrafish nacre/mitfa gene, expressed in all embryonic melanogenic cells, perturb only neural crest melanocytes, suggesting redundancy of mitfa with another gene in the zebrafish retinal pigment epithelium (RPE). Here, we describe a second zebrafish mitf gene, mitfb, which may fulfill this role. The proteins encoded by the two zebrafish mitf genes appear homologous to distinct isoforms generated by alternately spliced mRNAs of the single mammalian Mitf gene, suggesting specialization of the two zebrafish genes following a duplication event. Consistent with this hypothesis, expression of mitfa and mitfb is partially overlapping. mitfb is coexpressed with mitfa in the RPE at an appropriate time to compensate for loss of mitfa function in the nacre mutant but is not expressed in neural crest melanoblasts. Additionally, mitfb is expressed in the epiphysis and olfactory bulb where mitfa is not, and where Mitf expression has not previously been reported in other species. mitfb, but not a zebrafish ortholog of the closely related gene tfe3, can rescue neural crest melanophore development in nacre/mitfa mutant embryos when expressed via the mitfa promoter. These data suggest that mitfa and mitfb together may recapitulate the expression and functions of a single ancestral Mitf gene, and that mitfb may serve additional novel functions.

foxd3 encodes a winged helix/forkhead class transcription factor expressed in the premigratory neural crest cells of many vertebrates. We have investigated the function of this gene in zebrafish neural crest by a loss of function approach using antisense morpholino oligonucleotides and immunostaining for Foxd3 protein. Knockdown of Foxd3 expression produces deficits in several differentiated neural crest derivatives, including jaw cartilage, peripheral neurons, and glia, and iridophore pigment cells. Other derivatives, such as melanophore and xanthophore pigment cells are not affected. Reduction in the expression of several lineage-specific markers becomes evident soon after the onset of neural crest migration, suggesting that Foxd3 knockdown affects these lineages at early stages in their development. In contrast, analysis of the expression of early neural crest markers indicates little effect on neural crest induction or initial emigration. Finally, cell transplantation suggests that with respect to dorsal root ganglia neurons the Foxd3 requirement is cell autonomous, although Foxd3 itself is not detectable in differentiated DRG neurons. These results suggest that in zebrafish Foxd3 may not be required for induction of neural crest identity but is necessary for the differentiation of a subset of neural crest cell fates, perhaps in precursors of particular neural crest lineages.

We describe a mechanistic model whereby Foxd3, a forkhead transcription factor, prevents neural crest-derived precursors from acquiring a melanophore fate. Foxd3 regulates this fate choice by repressing the mitfa promoter in a subset of neural crest cells. mitfa is only expressed in a Foxd3-negative subset of neural crest cells, and foxd3 mutants show an increase in the spatial domain of mitfa expression, thereby suggesting Foxd3 limits the mitfa domain. Furthermore, foxd3:gfp transgenic zebrafish reveal foxd3 expression in xanthophore precursors and iridophores, but not in terminally differentiated melanophores. Luciferase experiments and embryo mRNA injections indicate Foxd3 acts directly on the mitfa promoter to negatively regulate mitfa expression. Taken together, our data suggests the presence of Foxd3 in a subset of precursors leads to mitfa repression and suppression of melanophore fate. MITF, the human mitfa ortholog, has recently been described as an oncogene and implicated in various forms of melanoma. Understanding the mechanisms that regulate mitfa and melanophore development could prove informative in the treatment and prevention of these human diseases.

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We report the isolation and identification of a new mutation affecting pigment cell fate in the zebrafish neural crest. Homozygous nacre (nac(w2)) mutants lack melanophores throughout development but have increased numbers of iridophores. The non-crest-derived retinal pigment epithelium is normal, suggesting that the mutation does not affect pigment synthesis per se. Expression of early melanoblast markers is absent in nacre mutants and transplant experiments suggested a cell-autonomous function in melanophores. We show that nac(w2) is a mutation in a zebrafish gene encoding a basic helix-loop-helix/leucine zipper transcription factor related to microphthalmia (Mitf), a gene known to be required for development of eye and crest pigment cells in the mouse. Transient expression of the wild-type nacre gene restored melanophore development in nacre(−/−) embryos. Furthermore, misexpression of nacre induced the formation of ectopic melanized cells and caused defects in eye development in wild-type and mutant embryos. These results demonstrate that melanophore development in fish and mammals shares a dependence on the nacre/Mitf transcription factor, but that proper development of the retinal pigment epithelium in the fish is not nacre-dependent, suggesting an evolutionary divergence in the function of this gene.

The mitfa gene encodes a zebrafish ortholog of the microphthalmia-associated transcription factor (Mitf) which, like its counterparts in other species, is absolutely required for development of neural crest melanocytes. In order to evaluate mitfa’s role in different stages of melanocyte development, we have identified hypomorphic alleles of mitfa, including two alleles that are temperature-sensitive for melanocyte development. Molecular analysis revealed that the mitffh53ts results from a single base pair change producing an asparagine to tyrosine amino acid substitution in the DNA-binding domain, and the mitfavc7 ts allele is a mutation in a splice donor site that reduces the level of correctly-spliced transcripts. Splicing in the mitfavc7 allele does not itself appear to be temperature-dependent. A third, hypomorphic allele, mitfaz25 results in an isoleucine to phenylalanine substitution in the first helix domain of the protein. Temperature upshift experiments with mitfafh53ts show that mitfa is required at several stages of melanocyte differentiation, including for expression of the early melanoblast marker dct, again for progression from dct expression to differentiation, and again for maintenance of dendritic form following differentiation. mitfafh53ts mutants recover melanocytes within 2–3 days when downshifted at all stages of larval development. However, when melanocyte stem cells (MSCs) are ablated by early treatment with the erbB3 inhibitor AG1478, melanocyte recovery is lost by 48 hours. This result indicates first that the MSC is established at the restrictive temperature, and that melanoblasts die or lose the ability to recover after being held at the restrictive temperature for approximately one day.