The Drosophila Pax-6 homologs eyeless (ey) and twin of eyeless (toy) are expressed
in the eyes and in the central nervous system (CNS). In addition to the pivotal functions
in eye development, previous studies revealed that ey also plays important roles in
axonal development of the mushroom bodies, centers for associative learning and memory.
It has been reported that a second intron enhancer that contains several Pax-6 binding
sites mainly controls the eye-specific expression, but the DNA sequences that control
CNS expression are unknown. In this work, we have dissected transcriptional enhancer
elements of the ey gene that are required for the CNS expression in various developmental
stages. We first show that CNS expression is independent of the eye-specific enhancer
of the second intron. By systematic reporter studies, we have identified several discrete
DNA elements in the 5' upstream region and in the second intron that cooperatively
interact to generate most of the ey expression pattern in the CNS. DNA sequence comparison
between the ey genes of distant Drosophila species has identified conserved modules
that might be bound by the upstream regulatory factors of the ey gene in CNS development.
Furthermore, by RNA interference and mutant studies, we show that ey expression in
the brain is independent of the activity of toy and ey itself whereas in the eye primordia
it requires both, supporting the notion that ey and toy are regulated by parallel
and independent regulatory cascades in brain development.