optix is involved in eyespot development via a possible positional information mechanism

Novel organismal traits might reuse ancestral gene-regulatory networks (GRNs) in their development, but data supporting this mechanism are still sparse. Here we show the reuse of an ancestral insect venation gene regulatory subnetwork patterning the sharp and distinct rings of color in butterfly eyespots. Using laser microdissection followed by RNA-Seq we first obtained transcriptional profiles of the anterior and posterior compartment of larval wings, and eyespot and adjacent control tissue in pupal wings of Bicyclus anynana butterflies. We identified key venation patterning genes such as Mothers against dpp 6 (Mad6), thickveins, Optix, spalt, optomotor-blind (omb), aristaless, cubitus interruptus, and patched differentially expressed (DE) across compartments, and a sub-set of these genes also DE across eyespot and non-eyespot samples. Fluorescent in-situ hybridization (HCR3.0) on the jointly DE genes Mad6, Optix, and spalt, as well as dpp showed clear eyespot-center, eyespot-rings, and compartment-specific expression. Knocking out dpp resulted in an individual with venation defects and loss of eyespots, whereas knockouts of Optix and spalt resulted in the loss of orange scales and black scales, respectively. Furthermore, using CRISPR-Cas9 followed by immunostainings, we showed that Spalt represses Optix in the central region of the eyespot, limiting Optix expression to a more peripheral ring, which parallels the regulatory interaction found in venation patterning in the anterior compartment of fly larval wings. These network similarities suggest that part of the venation GRN was co-opted to aid in the differentiation of the eyespot rings. One-sentence summaryWe showed the reuse of an ancestral insect wing venation GRN in patterning a novel complex trait in butterflies.