Shared regulatory networks link vein positioning and eyespot ring formation in butterflies

Novel traits might evolve via the repurposing of existing gene-regulatory networks. To demonstrate that a novel trait is the product of such co-option, we sought to show that the same genes and genetic interactions are taking place in different traits in a body. We examined the developmental specifications of two distinct traits that arose millions of years apart, wing veins and eyespot rings in butterflies. These two traits express a few genes in common along sharp boundaries that could derive from a shared conserved developmental program or through convergent evolution. Using laser-microdissected wing tissues followed by in-situ hybridization and antibody stainings we spatially mapped differentially expressed genes across those boundaries. We found that the expression domains of transcription factors Optix, optomotor-blind, and spalt in eyespot rings were similar to those observed in earlier vein positioning in butterflies and Drosophila. Furthermore, using CRISPR-Cas9 followed by immunostaining, we showed that Optix and spalt shared the same regulatory interaction. We propose that a primitive developmental program involved in vein positioning has been reused in the differentiation of the eyespot rings in nymphalid butterflies.