Dominant-negative form of the Pax6 homolog eyeless for tissue-specific loss-of-function studies in the developing eye and brain in drosophila.

Abstract

The Drosophila Pax6 homolog eyeless has essential roles in the development of the eye and brain in Drosophila (Quiring et al., 1994; Halder et al., 1995; Noveen et al., 2000; Kurusu et al., 2000; Callaerts et al., 2001). During eye development, eyeless is initially expressed in the embryonic eye anlagen and subsequently throughout the eye part of the eye-antennal imaginal disc in first and second instar larvae. Eventually, in third instar larvae, eyeless expression becomes restricted to the undifferentiated precursor cells anterior to the morphogenetic furrow in the eye disc. Mutations in eyeless result in increased apoptosis in the third instar eye disc, leading to partial or complete loss of eye structures. In the developing central nervous system, eyeless is continuously expressed in the ventral ganglion and brain, starting in the embryonic stages and throughout adulthood. In the adult brain, eyeless is expressed in the mushroom body neurons or Kenyon cells, in the medulla of the optic lobes, and in discrete cell populations in the central brain. Mutations in eyeless result in severely disrupted optic lobes, mushroom bodies, and central complex (Callaerts et al., 2001). Our understanding of the exact requirements for eyeless in the eye discs at various stages of larval development, as well as the role of eyeless in the different parts of the brain, would benefit greatly from clonal analyses. The location of the eyeless gene on chromosome 4, which lacks recombination and the absence of FRT chromosomes for FLP-recombinase-induced mitotic recombination, make clonal analysis at best tedious. Several studies in different species have made use of the repression domain of the Engrailed protein to generate dominant-negative versions of transcription factors and study their roles in development (e.g., Conlon et al., 1996; Gross and McClay, 2001; Glavic et al., 2001). We have generated transgenic lines harboring UAS-Eyeless-Engrailed Repression Domain-fusions (P{UAS.ey1-545.enRD}) (Fig. 1) to study the role of the eyeless gene in a spatially restricted or clonal fashion using the GAL4-UAS system. Expression of {UAS.ey1-545.enRD} using two different GAL4 lines with patterns overlapping with the wild-type eyeless expression pattern result in eye and brain phenotypes comparable to the phenotypes observed in homozygous eyeless mutants (see Fig. 2), thereby confirming that {UAS.ey1-545.enRD} acts as a dominant-negative version of eyeless. These reagents should prove useful for clonal analysis of the role of eyeless in the developing eye and brain.