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.

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 sizes^3-6. 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

1. Ramírez, F. et al. deepTools2: a next-generation web server for deep-sequencing data analysis. Nucleic Acids Res 44, W160–W165 (2016).

2. Thorvaldsdóttir, H., Robinson, J. T. & Mesirov, J. P. Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration. Brief Bioinform 14, 178–192 (2013).

3. Mazo-Vargas, A. et al. Macroevolutionary shifts of WntA function potentiate butterfly wing-pattern diversity. Proc. Natl. Acad. Sci. 114, 10701–10706 (2017).

4. Zhang, L. & Reed, R. D. Genome editing in butterflies reveals that spalt promotes and Distal-less represses eyespot colour patterns. Nat Commun 7, 11769 (2016).

5. Zhang, L., Mazo-Vargas, A. & Reed, R. D. Single master regulatory gene coordinates the evolution and development of butterfly color and iridescence. Proc. Natl. Acad. Sci. U. S. A. 114, 10707–10712 (2017).

6. Connahs, H. et al. Activation of butterfly eyespots by Distal-less is consistent with a reaction-diffusion process. Development 146, dev169367 (2019).

There are multiple files that contain either the dorsal or ventral side of the fore and hindwings. The files are labeled with the ID of the cis-regulatory element, followed by species ID (wcreDp, wcreAv, wcreHhim, wcreJc, wcreVc), then plate number (p1, p2, p3...), side (dorsal/ventral).tif, for example, "CRE03_wcreDp01_p1_Dorsal.tif". DataS1.txt includes information about the mutant clones. Location: DFw=Dorsal Forewing, DHw=Dorsal Hindwing, VFw= Ventral Forewing, VHw=Ventral Hindwing. The knock-out effect in adult wings is coded as Loss-of-Function (L), Gain-of-Function (G), presence of Loss-of-Function and Gain-of-Function effects on the same wing surface (T), and ambiguous effects (A). No phenotype effects (NP).