In Heliconius butterflies, wing pattern diversity is controlled by a few genes of large effect that regulate colour pattern switches between morphs and species across a large mimetic radiation. One of these genes, cortex, has been repeatedly associated with colour pattern evolution in butterflies. Here we carried out CRISPR knock-outs in multiple Heliconius species and show that cortex is a major determinant of scale cell identity. Chromatin accessibility profiling and introgression scans identified cis-regulatory regions associated with discrete phenotypic switches. CRISPR perturbation of these regions in black hindwing genotypes recreated a yellow bar, revealing their spatially limited activity. In the H. melpomene/timareta lineage, the candidate CRE from yellow-barred phenotype morphs is interrupted by a transposable element, suggesting that cis-regulatory structural variation underlies these mimetic adaptations. Our work shows that cortex functionally controls scale colour fate and that its cis-regulatory regions control a phenotypic switch in a modular and pattern-specific fashion.

CRISPR/Cas9 genome editing

Guide RNAs were designed corresponding to GGN₂₀NGG sites located within the cortex coding region using the program Geneious. To increase target specificity, guides were checked against an alignment of both melpomene and erato re-sequence data at the scaffolds containing the cortex gene, and selected based on sequence conservation across populations. Based on these criteria, each individual guide was checked against the corresponding genome for off-target effects, using the default Geneious algorithm. Guide RNAs with high conservation and low off-target scores were then synthesised following the protocol by Bassett and Liu, 2014 (https://doi.org/10.1016/j.ymeth.2014.02.019). Injections were performed following procedures described in Mazo-Vargas et al., 2017 (https://doi.org/10.1073/pnas.1708149114), within 1-4 hours of egg laying. Images deposited here were all imaged under the Keyence VHX-5000 digital microscope. 

Scanning Electron Microscopy (SEM) Imaging

Individual scales from wild type and mutant regions of interest were collected by brushing the surface of the wing with an eyelash tool, then dusted onto an SEM stub with double-sided carbon tape. Stubs were then colour imaged under the Keyence VHX-5000 microscope for registration of scale type. Samples were sputter-coated with one 12.5 nm layer of gold for improving sample conductivity. SEM images were acquired on a FEI Teneo LV SEM, using secondary electrons (SE) and an Everhart-Thornley detector (ETD) using a beam energy of 2.00 kV, beam current of 25 pA, and a 10 μs dwell time. Individual images were stitched using the Maps 3.10 software (ThermoFisher Scientific).

Morphometrics analysis

Morphometric measurements of scale widths and ridge distances were carried out on between 10 and 20 scales of each type, using a custom semi-automated R pipeline that derives ultrastructural parameters from large SEM images. Briefly, ridge spacing was assessed by Fourier transforming intensity traces of the ridges acquired from the FIJI software. Scale width was directly measured in FIJI by manually tracing a line, orthogonal to the ridges, at the section of maximal width. 

Immunohistochemistry and image analysis

Pupal wings were dissected around 60 to 70 h post pupation in PBS and fixed at room temperature with fix buffer (400 µl 4% paraformaldehyde, 600 µl PBS 2mM EGTA) for 30 min. Subsequent washes were done in wash buffer (0.1% Triton-X 100 in PBS) before blocking the wings at 4°C in block buffer (0.05 g Bovine Serum Albumin, 10 ml PBS 0.1% Triton-X 100). Wings were then incubated in primary antibodies against Cortex (1:200, monoclonal rabbit anti-Cortex) at 4°C overnight, washed and added in secondary antibody (1:500, donkey anti-rabbit lgG, AlexaFlour 555, ThermoFisher Scientific A-31572). Before mounting, wings were incubated in DAPI with 50% glycerol overnight and finally transferred to mounting medium (60% glycerol/ 40% PBS 2mM EGTA) for imaging.

Z-stacked 2-channelled confocal images were acquired using a Zeiss Cell Observer Spinning Disk Confocal microscope. Image processing was done using FIJI plugins Trainable Weka Segmentation and BioVoxxel.

TIF files: high-resolution SEM images of wild type and mutant scales and Keyence microscope images

psd: Same files as above provided in psd format

.raw: Images used in FIJI file format for scale measurement analyses

xsls files: nanomorphometric raw measurements

.R files: R scripts used in nanomorphometric statistics can be found at https://github.com/Hanliconius.