Pax6 genes encode evolutionarily highly conserved transcription factors that are 
required for eye and brain development. Despite the characterization of 
mutations in Pax6 homologs in a range of organisms, and despite functional 
studies, it remains unclear what the relative importance is of the various parts 
of the Pax6 protein. To address this, we have studied the Drosophila Pax6 
homolog eyeless. Specifically, we have generated new eyeless alleles, each with 
single missense mutations in one of the four domains of the protein. We show 
that these alleles result in abnormal eye and brain development while 
maintaining the OK107 eyeless GAL4 activity from which they were derived. We 
performed in vivo functional rescue experiments by expressing in an 
eyeless-specific pattern Eyeless proteins in which either the paired domain, the 
homeodomain, or the C-terminal domain was deleted. Rescue of the eye and brain 
phenotypes was only observed when full-length Eyeless was expressed, while all 
deletion constructs failed to rescue. These data, along with the phenotypes 
observed in the four newly characterized eyeless alleles, demonstrate the 
requirement for an intact Eyeless protein for normal Drosophila eye and brain 
development. They also suggest that some endogenous functions may be obscured in 
ectopic expression experiments.