Cortex cis-regulatory switches establish scale colour identity and pattern diversity inHeliconius

Abstract

22The wing patterns of butterflies are an excellent system with which to study phenotypic evolution. The 23 incredibly diverse patterns are generated from an array of pigmented scales on a largely two-24 dimensional surface, resulting in a visibly tractable system for studying the evolution of pigmentation. 25In Heliconius butterflies, much of this diversity is controlled by a few genes of large effect that regulate 26 pattern switches between races and species across a large mimetic radiation. One of these genescortex 27 -has been repeatedly mapped in association with colour pattern evolution in both Heliconius and other 28Lepidoptera, but we lack functional data supporting its role in modulating wing patterns. Here we 29 carried out CRISPR knock-outs in multiple Heliconius species and show that cortex is a major 30 determinant of scale cell identity. Mutant wing clones lacking cortex showed shifts in colour identity, 31 with melanic and red scales acquiring a yellow or white state. These homeotic transformations include 32 changes in both pigmentation and scale ultrastructure, suggesting that cortex acts during early stages of 33 scale cell fate specification rather than during the deployment of effector genes. In addition, mutant 34 clones were observed across the entire wing surface, contrasting with other known Heliconius mimicry 35 loci that act in specific patterns. Cortex is known as a cell-cycle regulator that modulates mitotic entry 36in Drosophila, and we found the Cortex protein to accumulate in the nuclei of the polyploid scale 37 building cells of the butterfly wing epithelium, speculatively suggesting a connection between scale cell 38 endocycling and colour identity. In summary, and while its molecular mode of action remains 39 mysterious, we conclude that cortex played key roles in the diversification of lepidopteran wing patterns 40 in part due to its switch-like effects in scale identity across the entire wing surface. 41 42 3

Many functionally connected loci foster adaptive diversification along a neotropical hybrid zone

“…The Optix-bound dome/wash promoter was not classified as an adaptive hub, indicating that while many loci associated with selection on Optix binding sites form adaptive hubs, strong candidate loci for color pattern-associated divergence are not limited to hub loci. dome has been previously implicated in multiple butterfly wing patterning roles, including yellow mimicry patterns in Heliconius (29,30). Specifically, the study of the presence or absence of a yellow hindwing bar phenotype in H. melpomene found a significant association between single-nucleotide polymorphisms (SNPs) at the dome/wash locus and the yellow bar pattern.…”

Reuse of an insect wing venation gene-regulatory subnetwork in patterning the eyespot rings of butterflies

“…cortex , a cell cycle regulator, plays a role in melanin synthesis in Biston betularia and Heliconius butterflies (Nadeau et al, 2016; van’t Hof et al, 2016). Loss of cortex affects black pigmentation and increase the extent of the upper lamina of the black scales (Livraghi et al, 2020). In sum, these experiments suggest that ommochromes and melanins such as dopa-melanin, dopamine-melanin, xanthommatin, and dihydroxanthommatin might be directly involved in polymerizing chitin in particular patterns inside a scale cell and in regulating scale morphology.…”

From Patterning Genes to Process: Unraveling the Gene Regulatory Networks That Pattern Heliconius Wings

“…However, the expression of the gene appears to be more complex than initially envisioned. Although previous expression studies using in situ hybridization have shown a strong correlation between the position of cortex mRNA in wing disks of fifth instar larvae and distal black pattern elements in the adult wings of H. numata and H. melpomene (Nadeau et al, 2016;Saenko et al, 2019), the color switches induced by cortex knockouts (KOs) do not seem to act in a pattern specific way in H. erato and H. melpomene (Livraghi et al, 2020). Instead, cortex KOs result in ectopic switches from melanic (Type II) and red/brown/orange (Type III) into white/yellow (Type I) scale cells across the whole surface of both wings in H. erato and H. melpomene.…”

“…In H. numata, variation around the region that contains cortex controls all variation in wing patterns and is responsible for differences in yellow, orange, and black pattern elements among sympatric color pattern morphs (Nadeau et al, 2016;Saenko et al, 2019). cortex KO results in the appearance of Type I scales across the wing surface of Heliconius butterflies (see Livraghi et al, 2020). The signaling ligand WntA is on chromosome 10 and variation around the locus is associated with differences in forewing band size, shape and position in H. erato and other Heliconius species.…”

“…Specifically, expression of fizzy-related in Drosophila follicle cells induces cell endocycling and results in polyploidy, and cortex has been shown to interact with the APC/C and downregulate cyclins during female meiosis (Schaeffer et al, 2004;Shcherbata et al, 2004;Pesin and Orr-Weaver, 2007;Swan and Schüpbach, 2007). The Heliconius ortholog also localizes to the nucleus in developing pupal wings, suggesting it may also be interacting with the APC/C in developing butterfly scales (Livraghi et al, 2020). Other work has shown a correlation between scale cell ploidy and pigmentation state where increased ploidy coincides with darker scales (Cho and Nijhout, 2013;Iwata and Otaki, 2016) and cortex may thus be modulating pigmentation state by varying endocycle rates across the wing.…”