Activation of butterfly eyespots by Distal-less is consistent with a reaction-diffusion process

Eyespots on the wings of nymphalid butterflies represent colorful examples of pattern formation, yet the developmental origins and mechanisms underlying eyespot center differentiation are still poorly understood. Using CRISPR-Cas9 we re-examine the function of Distal-less (Dll) as an activator or repressor of eyespots, a topic that remains controversial. We show that the phenotypic outcome of CRISPR mutations depends upon which specific exon is targeted. In Bicyclus anynana, exon 2 mutations are associated with both missing and ectopic eyespots, and also exon skipping. Exon 3 mutations, which do not lead to exon skipping, produce only null phenotypes, including missing eyespots, lighter wing coloration and loss of scales. Reaction-diffusion modeling of Dll function, using Wnt and Dpp as candidate morphogens, accurately replicates these complex crispant phenotypes. These results provide new insight into the function of Dll as a potential activator of eyespot development, scale growth and melanization, and suggest that the tuning of Dll expression levels can generate a diversity of eyespot phenotypes, including their appearance on the wing.