Knockout of a single Pax6 gene (toy but not ey) leads to compound eye deficiency and small head in honeybees.

The compound eyes are crucial to honeybees, playing pivotal roles in color recognition, orientation, localization, and navigation processes. The development of compound eyes is primarily mastered by an evolutionarily conserved transcription factor Pax6. In honeybees, there are two Pax6 homologs: ey and toy. To gain a deeper understanding of their functions, we knock out both homologs using CRISPR/Cas9 technology. Intriguingly, we observe that toy knockout mutants have smaller heads without compound eyes and exhibit brain atrophy, while ey knockout mutants develop normal compound eyes, most of which die before/during their metamorphosis from pupa to adult. By comparing the head transcriptomes of four stages (larva, prepupa, pupa, and adult) in toy-knockout mutants versus normal controls, we identify significantly perturbed genes related to DNA binding transcription factors, neuron differentiation, and insect visual primordium development. Additionally, we find the interaction network of toy in honeybees differs obviously from that of D. melanogaster. Our findings suggest the two Pax6 genes serve distinct functions in honeybees and toy takes over the central function of ey in master-regulating the development of honeybee compound eyes. This adds new evidence for breaking the simplified view that some of conservative developmental toolkit genes function as all-or-nothing master regulators.