From the formation of embryonic appendages to the color of wings: Conserved and novel roles of aristaless1 in butterfly development

Highly diverse butterfly wing patterns have emerged as a powerful system for understanding the genetic basis of phenotypic variation. While the genetic basis of this pattern variation is being clarified, the precise developmental pathways linking genotype to phenotype are not well understood. The gene aristaless, which plays a role in appendage patterning and extension, has been duplicated in Lepidoptera. One copy, aristaless1, has been shown to control a white/yellow color switch in the butterfly Heliconius cydno, suggesting a novel function associated with color patterning and pigmentation. We have investigated the developmental basis of al1 in embryos, larvae, and pupae using new antibodies, CRISPR/Cas9, RNAi, qPCR assays of downstream targets, and pharmacological manipulation of upstream activators. We found that Al1 is expressed at the tips of developing embryonic appendages consistent with its ancestral role. In wings, we observed Al1 accumulation within developing scale cells of white H. cydno during early pupation while yellow scale cells exhibit little Al1 at this time point. Reduced Al1 expression is also associated with yellow scale development in al1 knockouts/knockdowns. Our work as a whole proposes a model in which high levels of Al1 during early pupation, are important for melanic pigmentation and specifying white portions of the wing while reduced levels of Al1 during early pupation promote upregulation of proteins needed to move and synthesize 3-OHK, promoting yellow pigmentation. This complementary research between ancestral and novel roles of aristaless1 allows us to discuss cellular mechanisms in the context of novel gene function.