Pax6 homologs are required for patterning both visual systems of the daddy-longlegs Phalangium opilio

The evolution of visual systems has compelled numerous investigations of developmental processes underlying eye patterning across Bilateria. It is well-established that homologs of the transcription factor Pax6 play a highly conserved role in eye fate specification and are at the top of the retinal determination gene network (RDGN) hierarchy. Curiously, Pax6 homologs do not appear to maintain this function in well-studied chelicerate models. It was recently proposed that the gene Pax2 (shaven; sv) may have subsumed the role of Pax6 in eye fate specification in chelicerates. However, no functional data are available for any chelicerate Pax homologs. We examined the incidence of Pax family genes across chelicerate phylogeny, and interrogated the expression and function of Pax2 and Pax6 homologs in the daddy-longlegs Phalangium opilio, which bears a highly plesiomorphic arrangement of visual systems. We show that the Pax6 homologs eyeless (ey) and twin of eyeless (toy) are expressed early in the developing head lobes of P. opilio, whereas sv is not. Gene silencing of ey, toy, and sv individually had no discernible effect on eye development. By contrast, double knockdown of ey and toy resulted in defects or loss in all three eye pairs of P. opilio. These data are consistent with a conserved role for Pax6 homologs in patterning both median and lateral visual systems across arthropods. Our results suggest that heterochronic shifts in expression, rather than changes in function, underlie the atypical dynamics of Pax genes in derived arachnid groups such as spiders.