10.5061/DRYAD.8GTHT76M0
Livraghi, Luca
0000-0002-2597-7550
University of Cambridge
Hanly, Joseph J.
0000-0002-9459-9776
George Washington University
Loh, Ling Sheng
0000-0003-0981-7984
George Washington University
Ren, Anna
0000-0001-9525-9661
George Washington University
Warren, Ian A.
University of Cambridge
Concha, Carolina
Smithsonian Tropical Research Institute
Wright, Charlotte
0000-0002-3971-4372
University of Cambridge
Walker, Jonah M.
University of Cambridge
Foley, Jessica
Smithsonian Tropical Research Institute
Arenas-Castro, Henry
Smithsonian Tropical Research Institute
Martin, Arnaud
0000-0002-5980-2249
George Washington University
McMillan, William O.
Smithsonian Tropical Research Institute
Jiggins, Chris D.
University of Cambridge
Van Bellghem, Steven M.
University of Puerto Rico System
Montejo-Kovacevich, Gabriela
0000-0003-3716-9929
University of Cambridge
Lewis, James J.
Cornell University
Perry, Micheal W.
University of California, San Diego
Goldberg, Zachary H.
0000-0003-2972-9682
University of California, San Diego
Lopez, Laura H.
University of Cambridge
Papa, Riccardo
University of Puerto Rico System
van der Heijden, Eva S.M.
University of Cambridge
Cortex cis-regulatory switches establish scale colour identity and pattern
diversity in Heliconius
Dryad
dataset
2020
Biotechnology and Biological Sciences Research Council
https://ror.org/00cwqg982
BB/R007500/1
National Science Foundation
https://ror.org/021nxhr62
IOS-1656553
National Science Foundation
https://ror.org/021nxhr62
IOS-1755329
Wellcome Trust
https://ror.org/029chgv08
Smithsonian Institution
https://ror.org/01pp8nd67
2021-07-18T00:00:00Z
2021-07-18T00:00:00Z
en
https://github.com/Hanliconius
https://doi.org/10.1101/2020.05.26.116533
https://doi.org/10.7554/eLife.68549
13890699982 bytes
5
CC0 1.0 Universal (CC0 1.0) Public Domain Dedication
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.