Arthropod <i>Pax</i> Gene Evolution: A Role for <i>Vanessa Cardui Twin of Eyeless</i> in Eye Development

Pax genes encode transcription factors involved in diverse processes. First identified in Drosophila, they have been found throughout the animal kingdom, suggesting highly conserved functions. Pax proteins are defined by a DNA-binding PRD domain along with variable presence of a homeodomain and octapeptide motif. Although some Pax genes have been studied in selected arthropod groups, less is known about phylogenetic relationships among arthropod Pax genes. Here, we analyzed their distribution and classification and established the painted lady butterfly, Vanessa cardui, to study Pax gene expression and function. Our phylogenetic analysis grouped arthropod Pax genes into 6 clades: Pax3/7, Pax1/9, Poxn, Pax6-like/eyg, Pax2/5/8, and Pax4/6. This large-scale analysis confirmed that the Pax3/7 gene paired was lost in Lepidoptera, which retain other Pax3/7 family members as well as all other Pax groups. Expression of Vcar-Pax genes during Vanessa embryonic development was largely similar to previous reports for Drosophila. To test functional conservation, we focused on the eye development master regulator, twin of eyeless (toy). Vcar-toy was expressed in the head lobes and embryonic RNA interference resulted in loss of larval eyes. In addition, Vcar-ey, a candidate downstream target of toy, was downregulated after Vcar-toy knockdown. Ectopic expression of Vcar-toy in Drosophila resulted in ectopic, Drosophila-like adult eyes, supporting the notion that gene regulatory networks regulating larval and adult eyes are conserved and also shared between Vanessa and Drosophila. Overall, these results suggest that Pax genes are highly conserved in arthropods and provide a butterfly model to study eye regulatory networks in Lepidoptera.