Reviewer #1 (Public Review):
In this paper the authors associate genetic variation in regulatory sequences of the gene cortex with the presence/absence of a yellow band of color in the wings of two species of Heliconius butterflies. They show that cortex is spatially regulated in larval wings, but the expression of this gene does not correlate with the presence or absence of the yellow band. Then they show that the gene is expressed in the nuclei of all cells of the pupal wing. By disrupting cortex they show that black cells (Type II) become white or yellow (Type I), and red scales (Type III) become paler across the whole wing.
By examining open regions of chromatin around cortex, they discover that at least in one of the species, the insertion of two transposable elements in an open region of chromatin associates with the presence of the yellow band. They show that disrupting this regulatory region in a race of butterflies that does not contain the yellow band, nor the TE insertions, leads to the loss of the black color in a band-like shape, and the appearance of yellow scales in that region of the wing. They identify a different region of open-chromatin in the other Heliconius species that when disrupted also leads to the transformation of black scales into yellow scales in a band-like pattern.
The authors achieved their aims and the results support their conclusions.
The strength of this manuscript lies in the use of multiple approaches to identify the likely causal genetic variation in the cortex locus that is responsible for the presence/absence of the yellow band. The only weakness (if I can call it that) is that it is still not clear how cortex, which is also expressed in the nuclei of the yellow scales in races that supposedly have the TE insertion and closed chromatin in that enhancer region, fail to develop black scales in that region of the wing.
This is one of the first few papers that examines the function of specific open regions of chromatin in the DNA of butterfly species using CRISPR-Cas9. The main novelty of this paper is in identifying how a gene with a homogeneous expression pattern across the wing (during the pupal stage) can still have "hidden" modular regulatory regions that drive unique functions (albeit not expression) is specific regions of the wing.
This work reminds me of the regulation of the vestigial gene in the wings of Drosophila. vestigial also has homogeneous expression across the wing pouch but it achieves this homogeneous expression via two separate enhancers that have complementary expression patterns.