Disrupting differentDistal-lessexons leads to ectopic and missing eyespots accurately modeled by reaction-diffusion mechanisms

Eyespots on the wings of nymphalid butterflies represent colorful examples of the process of pattern formation, yet the developmental origins and the mechanisms behind eyespot differentiation are still not fully understood. Here we re-examine the function of Distal-less (Dll) in eyespot development, which is still unclear. We show that CRISPR-Cas9 induced exon 2 mutations in Bicyclus anynana leads to exon skipping and ectopic eyespots on the wing. Exon 3 mutations, however, lead to null/missense transcripts, missing eyespots, lighter wing coloration, loss of scales, and a variety of other phenotypes implicating Dll in the process of eyespot differentiation. Reaction-diffusion modeling enabled exploration of the function of Dll in eyespot formation, and accurately replicated a wide-range of mutant phenotypes. These results confirm that Dll is a required activator of eyespot development, scale growth and melanization and point to a new mechanism of alternative splicing to achieve Dll over-expression phenotypes.