In the 1920s, biologists proposed that butterfly wing pattern diversity evolved as variations of a ground plan of pattern elements that vary in color, shape, and position between different species. In new research, George Washington University scientist Anyi Mazo-Vargas and colleagues found that major aspects of this ground plan are determined by an ancient array of ‘junk’ or non-coding regulatory DNA.
Danaus plexippus. Image credit: Gyulche1.
“We are interested to know how the same gene can build these very different looking butterflies,” Dr. Mazo-Vargas said.
“We see that there’s a very conserved group of switches non-coding DNA that are working in different positions and are activated and driving the gene.”
Previous studies uncovered key color pattern genes: one (WntA) that controls stripes and another (Optix) that controls color and iridescence in butterfly wings.
When Dr. Mazo-Vargas and co-authors disabled the Optix gene, the wings appeared black, and when the WntA gene was deleted, stripe patterns disappeared.
In the new study, they focused on the effect of non-coding DNA on the WntA gene.
Specifically, they ran experiments on 46 of these non-coding elements in five species of nymphalid butterflies, which is the largest family of butterflies: Junonia coenia, Vanessa cardui, Heliconius himera, Agraulis vanillae and Danaus plexippus.
In order for these non-coding regulatory elements to control genes, tightly wound coils of DNA become unspooled, a sign that a regulatory element is interacting with a gene to activate it, or in some cases, turn it off.
The researchers used a technology called assay for transposase-accessible chromatin using sequencing (ATAC-seq) to identify regions in the genome where this unraveling is occurring.
They compared ATAC-seq profiles from the wings of five butterfly species, in order to identify genetic regions involved in wing pattern development.
They were surprised to find that a large number of regulatory regions were shared across very different butterfly species.
The team then employed CRISPR-Cas gene editing technology to disable 46 regulatory elements one at a time, in order to see the effects on wing patterns when each of these non-coding DNA sequences were broken.
When deleted, each non-coding element changed an aspect of the wing patterns of the butterflies.
The scientists found that across Junonia coenia, Vanessa cardui, Heliconius himera and Agraulis vanillae, each of these non-coding elements had similar functions with respect to the WntA gene, proving they were ancient and conserved, likely originating in a distant common ancestor.
They also found that Danaus plexippus used different regulatory elements from the other four species to control its WntA gene, perhaps because it lost some of its genetic information over its history and had to reinvent its own regulatory system to develop its unique color patterns.
“We have progressively come to understand that most evolution occurs because of mutations in these non-coding regions,” said Cornell University’s Professor Robert Reed.
“What I hope is that this paper will be a case study that shows how people can use this combination of ATAC-seq and CRISPR to begin to interrogate these interesting regions in their own study systems, whether they work on birds or flies or worms.”
The results were published in the journal Science.
_____
Anyi Mazo-Vargas et al. 2022. Deep cis-regulatory homology of the butterfly wing pattern ground plan. Science 378 (6617): 304-308; doi: 10.1126/science.abi9407
