Fishes of the genus Danio exhibit diverse pigment patterns that serve as useful models for understanding the genes and cell behaviors underlying the evolution of adult form. Among these species, zebrafish D. rerio exhibit several dark stripes of melanophores with sparse iridophores that alternate with light interstripes of dense iridophores and xanthophores. By contrast, the closely related species D. nigrofasciatus has an attenuated pattern with fewer melanophores, stripes and interstripes. Here we demonstrate species differences in iridophore development that presage the fully formed patterns. Using genetic and transgenic approaches we identify the secreted peptide Endothelin-3 (Edn3)—a known melanogenic factor of tetrapods—as contributing to reduced iridophore proliferation and fewer stripes and interstripes in D. nigrofasciatus. We further show the locus encoding this factor is expressed at lower levels in D. nigrofasciatus owing to cis-regulatory differences between species. Finally, we show that functions of two paralogous loci encoding Edn3 have been partitioned between skin and non-skin iridophores. Our findings reveal genetic and cellular mechanisms contributing to pattern differences between these species and suggest a model for evolutionary changes in Edn3 requirements for pigment patterning and its diversification across vertebrates.

The molecular genetic toolkit of the Mexican axolotl, a classic model organism, has matured to the point where it is now possible to identify genes for mutant phenotypes. We used a positional cloning–candidate gene approach to identify molecular bases for two historic axolotl pigment phenotypes: white and albino. White (d/d) mutants have defects in pigment cell morphogenesis and differentiation, whereas albino (a/a) mutants lack melanin. We identified in white mutants a transcriptional defect in endothelin 3 (edn3), encoding a peptide factor that promotes pigment cell migration and differentiation in other vertebrates. Transgenic restoration of Edn3 expression rescued the homozygous white mutant phenotype. We mapped the albino locus to tyrosinase (tyr) and identified polymorphisms shared between the albino allele (tyr a) and tyr alleles in a Minnesota population of tiger salamanders from which the albino trait was introgressed. tyr a has a 142 bp deletion and similar engineered alleles recapitulated the albino phenotype. Finally, we show that historical introgression of tyr a significantly altered genomic composition of the laboratory axolotl, yielding a distinct, hybrid strain of ambystomatid salamander. Our results demonstrate the feasibility of identifying genes for traits in the laboratory Mexican axolotl.

Summary Teleosts comprise about half of all vertebrate species and exhibit an extraordinary diversity of adult pigment patterns that function in shoaling, camouflage and mate choice and have played important roles in speciation. Here, we review recent studies that have identified several distinct neural crest lineages, with distinct genetic requirements, that give rise to adult pigment cells in fishes. These lineages include post-embryonic, peripheral nerve associated stem cells that generate black melanophores and iridescent iridophores, cells derived directly from embryonic neural crest cells that generate yellow-orange xanthophores, and bipotent stem cells that generate both melanophores and xanthophores. This complexity in adult chromatophore lineages has implications for our understanding of adult traits, melanoma, and the evolutionary diversification of pigment cell lineages and patterns.

31Fishes of the genus Danio exhibit diverse pigment patterns that serve as useful models for 32 understanding the genes and cell behaviors underlying the evolution of adult form. Among these 33 species, zebrafish D. rerio exhibit several dark stripes of melanophores with sparse iridophores 34 that alternate with light interstripes of dense iridophores and xanthophores. By contrast, the 35 closely related species D. nigrofasciatus has an attenuated pattern with fewer melanophores, 36 stripes and interstripes. Here we demonstrate species differences in iridophore development 37 that presage the fully formed patterns. Using genetic and transgenic approaches we identify the 38 secreted peptide Endothelin-3 (Edn3)-a known melanogenic factor of tetrapods-as 39 contributing to reduced iridophore proliferation and fewer stripes and interstripes in D. 40nigrofasciatus. We further show the locus encoding this factor is expressed at lower levels in D. 41nigrofasciatus owing to cis-regulatory differences between species. Finally, we show that 42 functions of two paralogous loci encoding Edn3 have been partitioned between skin and non-43 skin iridophores. Our findings reveal genetic and cellular mechanisms contributing to pattern 44 differences between these species and suggest a model for evolutionary changes in Edn3 45 requirements across vertebrates. 47Author Summary 48Neural crest derived pigment cells generate the spectacular variation in skin pigment patterns 49 among vertebrates. Mammals and birds have just a single skin pigment cell, the melanocyte, 50 whereas ectothermic vertebrates have several pigment cells including melanophores, 51 iridophores and xanthophores, that together organize into a diverse array of patterns. In the 52 teleost zebrafish, Danio rerio, an adult pattern of stripes depends on interactions between 53 pigment cell classes and between pigment cells and their tissue environment. The close 54 relative, D. nigrofasciatus has fewer stripes and prior analyses suggested a difference between 55 these species that lies extrinsic to the pigment cells themselves. A candidate for mediating this 56 difference is Endothelin-3 (Edn3), essential for melanocyte development in warm-blooded 57 animals, and required by all three classes of pigment cells in an amphibian. We show that Edn3 58 specifically promotes iridophore development in Danio, and that differences in Edn3 expression 59 contribute to differences in iridophore complements, and striping, between D. rerio and D. 60 nigrofasciatus. Our study reveals a novel function for Edn3 and provides new insights into how 61 changes in gene expression yield morphogenetic outcomes to effect diversification of adult 62 form. 65Quantitative genetic analyses and association studies have made progress in identifying loci, 66 and even specific nucleotides, that contribute to morphological differences between closely 67 related species and strains. Yet it remains often mysterious how allelic effects are translated 68 into specific cellular outcomes of differentiation and...