The Pax6 genes eyeless and twin of eyeless are required for global patterning of the ocular segment in the Tribolium embryo

The transcription factor gene Pax6 is widely considered a master regulator of eye development in bilaterian animals. However, the existence of visual organs that develop without Pax6 input and the considerable pleiotropy of Pax6 outside the visual system dictate further studies into defining ancestral functions of this important regulator. Previous work has shown that the combinatorial knockdown of the insect Pax6 orthologs eyeless (ey) and twin of eyeless (toy) perturbs the development of the visual system but also other areas of the larval head in the red flour beetle Tribolium castaneum. To elucidate the role of Pax6 during Tribolium head development in more detail, we studied head cuticle morphology, brain anatomy, embryonic head morphogenesis, and developmental marker gene expression in combinatorial ey and toy knockdown animals. Our experiments reveal that Pax6 is broadly required for patterning the anterior embryonic head. One of the earliest detectable roles is the formation of the embryonic head lobes, which originate from within the ocular segment and give rise to large parts of the supraesophageal brain including the mushroom body, a part of the posterior head capsule cuticle, and the visual system. We present further evidence that toy continues to be required for the development of the larval eyes after formation of the embryonic head lobes in cooperation with the eye developmental transcription factor dachshund (dac). The sum of our findings suggests that Pax6 functions as a competence factor throughout the development of the insect ocular segment. Comparative evidence identifies this function as an ancestral aspect of bilaterian head development.