Mushroom bodies (MBs) are the centers for olfactory associative learning and elementary cognitive functions in the Drosophila brain. By high-resolution neuroanatomy, we show that eyeless (ey), twin of eyeless, and dachshund (dac), which are implicated in eye development, also are expressed in the developing MBs. Mutations of ey completely disrupted the MB neuropils, and a null mutation of dac resulted in marked disruption and aberrant axonal projections. Genetic analyses demonstrated that, whereas ey and dac synergistically control the structural development of the MBs, the two genes are regulated independently in the course of MB development. These data argue for a distinct combinatorial code of regulatory genes for MBs as compared with eye development and suggest conserved roles of Pax6 homologs in the genetic programs of the olfactory learning centers of complex brains.

The molecular mechanisms of head development are a central question in vertebrate and invertebrate developmental biology. The anteriorly expressed homeobox gene otd in Drosophila and its homolog Otx in mouse are required for the early development of the most anterior part of the body, suggesting that a fundamental genetic program of cephalic development might be conserved between vertebrates and invertebrates. We have examined this hypothesis by introducing the human Otx genes into f lies. By inducing expression of the human Otx homologs with a heat shock promoter, we found that both Otx1 and Otx2 functionally complement the cephalic defects of a f ly otd mutant through specific activation and inactivation of downstream genes. Combined with previous morphological studies, these results are consistent with the view that a common molecular ground plan of cephalization was invented before the diversification of the protostome and the deuterostome in the course of metazoan evolution.The developmental mechanisms and the evolution of the vertebrate and invertebrate cephalic structures have been debated extensively since the last century (1, 2). Based on anatomical and paleontological observations, it traditionally has been accepted that vertebrate and invertebrate head and brain were invented independently. Although this notion still is contested intensively, recent molecular and developmental studies have revealed a common mechanism of axial patterning for the trunk regions of protostomes and deuterostomes. A striking example is seen in the conserved colinearity between the expression patterns along the antero-posterior axis and the chromosomal locations of the Drosophila homeotic (HOM) complex genes and the vertebrate Hox complex genes (3-5). It also has been shown that the molecular mechanisms underlying dorsoventral patterning are conserved between flies and vertebrates (6).The basic organizations of the insect and the vertebrate heads have been studied intensively in the past years. The developing vertebrate brains are proposed to be organized in distinct subregions that reflect a basic metameric regionalization (7,8). Similarly, morphological and developmental studies on insect brains reveal an underlying metameric organization (9-11). Among the regulatory genes that control cephalic development, the anteriorly expressed homeobox-containing gene orthodenticle (otd) in Drosophila (12, 13) and its homolog Otx in mice (14-19) play important roles in early patterning of the most anterior parts of the body, suggesting that fundamental genetic programs of cephalic development might be conserved (20,21,22). Here, we show that, despite the evolutionary distance and sequence diversity, the human Otx genes rescue developmental cephalic defects of a Drosophila otd mutant, indicating that the human Otx genes are functional homologs of the Drosophila gene. Our findings suggest that the basic regulatory interactions controlling the cephalic development may be conserved between the flies and mammals and are consistent ...