Deep cis-regulatory homology of the butterfly wing pattern groundplan

Butterfly wing patterns derive from a deeply conserved developmental groundplan, yet are highly diverse and evolve rapidly. It is poorly understood how gene regulatory architectures can accommodate both deep homology and adaptive change. To address this, we characterized the cis-regulatory evolution of the groundplan gene WntA in nymphalid butterflies. Comparative ATAC-seq and in vivo knockouts of 46 Cis-Regulatory Elements (CREs) across five species revealed extensive sequence homology of groundplan CREs, except in monarch butterflies. Most CRE perturbation assays showed effects spanning multiple elements and encoding both positive and negative regulatory functionality. Our results provide little support for models predicting rapid turnover of single-trait enhancers and suggest morphological change within a tissue is achieved by tuning deeply conserved and highly sensitive networks of interdependent CREs.

Methods
Candidate regulatory elements were identified, ATAC-seq signal from heads was subtracted from wings using bamCompare1 and inspected in the IGV browser2. We restricted our focus area to the WntA TAD and selected sharp, differentially accessible peaks, with different conservation levels, for CRISPR validation. We designed between 2–4 sgRNAs per peak, following the motif N20NGG, and purchased sgRNAs from Synthego. G0 adult wings were screened for mosaic color pattern mutations. An individual with a successful mutation can present clones with different deletion alleles of different sizes3-6. To "call" a mutant, we look for asymmetric disruptions in the patterns between left and right wings since asymmetry precludes natural variation.  Wings presenting mosaic mutant clones were scanned, dorsal and ventrally, using an EPSON Perfection V500 scanner, with 800 dpi (Dots Per Inch).

References

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