Reuse of an insect wing venation gene-regulatory subnetwork in patterning the eyespot rings of butterflies

Abstract

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 anynanabutterflies. We identified key venation patterning genes such as Mothers against dpp 6 (Mad6), thickveins, Optix, spalt, optomotor-blind (omb), aristaless, cubitus interruptus, and patcheddifferentially expressed (DE) across compartments, and a sub-set of these genes also DE across eyespot and non-eyespot samples. Fluorescent in-situhybridization (HCR3.0) on the jointly DE genes Mad6, Optix,and spalt, as well as dppshowed clear eyespot-center, eyespot-rings, and compartment-specific expression. Knocking out dppresulted in an individual with venation defects and loss of eyespots, whereas knockouts of Optixand spaltresulted in the loss of orange scales and black scales, respectively. Furthermore, using CRISPR-Cas9 followed by immunostainings, we showed that Spalt represses Optixin the central region of the eyespot, limiting Optixexpression 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 summary

We showed the reuse of an ancestral insect wing venation GRN in patterning a novel complex trait in butterflies.