Pax6 genes encode transcription factors with two DNA-binding domains that are 
highly conserved during evolution. In Drosophila, two Pax6 genes function in a 
pathway in which twin of eyeless (toy) directly regulates eyeless (ey), which is 
necessary for initiating the eye developmental pathway. To investigate the gene 
duplication of Pax6 that occurred in holometabolous insects like Drosophila and 
silkworm, we used different truncated forms of toy and small eyes (sey), and 
tested their capacity to induce ectopic eye development in an ey-independent 
manner. Even though the Paired domains of TOY and SEY have DNA-binding 
properties that differ from those of the Paired domain of EY, they all are 
capable of inducing ectopic eye development in an ey mutant background. We also 
show that one of the main functional differences between toy and ey lies in the 
C-terminal region of their protein products, implying differences in their 
transactivation potential. Furthermore, we show that only the homeodomain (HD) 
of EY is able to downregulate the expression of Distal-less (Dll), a feature 
that is required during endogenous eye development. These results suggest 
distinct functions of the two DNA-binding domains of TOY and EY, and significant 
evolutionary divergence between the two Drosophila Pax6 genes.