Regulatory changes underlying the evolution of skylight navigation

Polarized skylight is an ancient navigational cue for many animals. In insects, its detection relies on the dorsal rim area (DRA), a specialized region of the compound eye. DRAs are widespread and vary in morphology and spectral tuning, raising the question of how this diversity evolved. Here we show that the DRA expression of the Drosophila transcription factor homothorax (encoded by the gene hth) depends on a 675 bp minimal enhancer. Genome editing of this enhancer abolishes both DRA formation and polarization-guided behavior. Furthermore, mutagenesis of conserved motifs suggests that enhancer activity is regulated by cooperative transcription factor activity. Newly generated antibodies from honeybees, butterflies, and mosquitoes reveal no hth expression in their DRAs, whereas hth is expressed across higher flies (Brachycera), coinciding with the loss of its homolog Pknox/Prep1. Phylogenetic comparisons therefore suggest that hth's role in DRA specification arose relatively recently, coinciding with evolutionary changes in photoreceptor optics and photochemistry from lower to higher flies.