Genome editing in butterflies reveals that spalt promotes and Distal-less represses eyespot colour patterns

Abstract

Butterfly eyespot colour patterns are a key example of how a novel trait can appear in association with the co-option of developmental patterning genes. Little is known, however, about how, or even whether, co-opted genes function in eyespot development. Here we use CRISPR/Cas9 genome editing to determine the roles of two co-opted transcription factors that are expressed during early eyespot determination. We found that deletions in a single gene, spalt, are sufficient to reduce or completely delete eyespot colour patterns, thus demonstrating a positive regulatory role for this gene in eyespot determination. Conversely, and contrary to previous predictions, deletions in Distal-less (Dll) result in an increase in the size and number of eyespots, illustrating a repressive role for this gene in eyespot development. Altogether our results show that the presence, absence and shape of butterfly eyespots can be controlled by the activity of two co-opted transcription factors.

A Practical Guide to CRISPR/Cas9 Genome Editing in Lepidoptera

“…Our results show that donor DNA with 500 bp homology arms is sufficient for precise in-frame knockins. Compared to NHEJ-mediated high efficiency knockouts (69% in the case of V. cardui Ddc deletion knockouts (Zhang and Reed 2016)), the rate of HDR-mediated targeted integration is low, at~3% in our most recent trials. It has been shown that knocking out factors in the NHEJ pathway can enhance the HDR pathway and increase gene targeting efficiency in Bombyx (Ma et al 2014;Zhu et al 2015).…”

“…This was an important case study because it showed that the general approach used by Wang et al (2013) in B. mori could be transferred to other species and still retain the same level of high efficiency. Two more notable technical advancements include the production of an 18 kb deletion in B. mori by Zhang et al (2015) the longest deletion we know of in Lepidoptera, and much longer than anything in (Zhang and Reed 2016;Perry et al 2016), which was an important improvement to the protocol that significantly simplifies the genome editing workflow. Through our lab's research on butterfly wing pattern development, we have tried most of the methods described in the studies cited above, and we have gained significant experience in porting these protocols across species.…”

“…8.1a in Zhang and Reed, 2016). Zhang Z et al (2015) The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material.…”

“…This protocol includes a specific example of the Reed Lab's work deleting the melanin pigmentation pathway gene Ddc as previously reported (Zhang and Reed 2016).…”

Molecular Mechanisms Underlying Color Vision and Color Formation in Dragonflies

“…Molecular bases underlying color pattern formation and its evolution in dragonflies are just as fascinating and challenging as in butterflies. Recently, effective RNAi-and genome-editing methods have been developed for gene functional analyses in butterflies (Ando and Fujiwara 2013;Nishikawa et al 2015;Li et al 2015;Perry et al 2016;Zhang and Reed 2016;Beldade and Peralta 2017). Applying these methods to dragonflies will be an important step toward future studies in this field (Okude et al 2017).…”