DOI: 10.5061/dryad.8cz8w9gpr
title: Deep cis-regulatory homology of the butterfly wing pattern groundplan

description: 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.
description: Candidate regulatory elements were identified, ATAC-seq signal
 from heads was subtracted from wings using
 bamCompare<sup>1</sup> and inspected in the IGV
 browser<sup>2</sup>. We restricted our focus area to the
 <em>WntA</em> 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
 N<sub>20</sub>NGG, 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<sup>3-6</sup>. 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).
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description: 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).