Evolution of Endothelin Signaling and Diversification of Adult Pigment Patterns

Abstract

Vertebrate forms are tremendously diverse. While there are many studies identifying genetic loci that have played a role in diversification, there are fewer that elucidate specific evolutionary changes and mechanisms at the cellular level which drive differences in adult morphology. Pigmentation is an especially tractable trait to study mechanisms of diversification in form. Fishes of the genus Danio exhibit diverse pigment patterns that serve as useful models for understanding the genes and cell behaviors underlying 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 set-up 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, and that functions of two paralogous loci encoding Edn3 have been partitioned between skin and non-skin iridophores. In contrast, Edn3 is required by all three pigment cell types in the developmental model the Mexican axolotl (Ambystoma mexicanum), suggesting a model for evolutionary changes in Edn3 requirements in pigment pattern diversification across vertebrates. We show that the locus responsible for the historic axolotl pigment phenotype, “white” (d/d), is edn3. Transgenic restoration of Edn3 expression in the white axolotl rescues the pigmentation phenotype, while knockdown of Edn3 in wild-type axolotls via morpholino injections phenocopies white.