PMC

The C-terminal domain of Eyeless functions as a transcriptional activator. Luciferase activity relative to pRmGVP16 for four protein fusions with the GAL4 DNA-binding domain. pRmG: empty vector negative control encoding the GAL4 DNA-binding domain only. pRmGEy: Eyeless C-terminal domain fusion. pRmGEy-D1Da: Eyeless C-terminal domain fusion with the eyD1Da mutation. pRmGVP16: Fusion with the strong transactivation domain of Herpes simplex VP16 as positive control. pRmGEy is significantly different from pRmGEy-D1Da and pRmG (p=0.0001, Duncan test).

Abnormal subcellular localization of protein encoded by the allele ey^OK107/6. (A-C) Eyeless localizes to the nucleus in wild type eye discs. Single channel image of wildtype eye discs in a heterozygous Pabp2^CC00380 background, which ubiquitously expresses a nuclear-localized Pabp2-GFP fusion protein, stained with Eyeless (A) or GFP (B). (C) Overlay of Eyeless (magenta) and GFP (green) reveals nuclear colocalization of Pabp2-GFP and Eyeless. (D-F) Eyeless is primarily cytoplasmic in ey^OK107/6 mutant eye discs. Single channel image stained with Eyeless (D) or GFP (E). (F) Overlay of Eyeless (magenta) and GFP (green) reveals limited overlap between Pabp2-GFP and Eyeless and thus cytoplasmic localization of mutant Eyeless protein. Size bars are 5 μm.

(A) Schematic representation of the predicted protein products encoded by wild type eyeless, the four ey^OK107/X alleles (stars indicate position of the amino acid substitution; this paper), ey^D1Da and ey^JD (), and ey^EH and ey¹¹ (). The Eyeless protein consists of the paired domain (PRD, black shading), the linker region (LR), the homeodomain (HD, grey shading), and the C-terminal domain (CTD). (B) Head and eye defects in homozygous ey^OK107/X mutants. (C) Anti-Fasciclin II antibody staining reveals α, β and γ lobes of the mushroom bodies. One of the frequently observed phenotypes is the loss of lobes (marked with arrowheads in the different panels) as was previously documented in . Size bars are 25 μm.

The eye, head, and brain phenotypes observed in homozygous eyeless flies can be rescued only by full-length Eyeless (UE10 = UAS full-length Eyeless) when driven by the Gal4 activity present in the ey^OK107/16 mutant. Eyeless UAS constructs in which one domain of the Eyeless protein has been removed do not result in a phenotypic rescue in the same mutant background (see for complete results). (A-D) Anti-Fasciclin II antibody staining reveals α, β and γ lobes of the mushroom bodies. Note the lack of lobes in C and D (marked as an outline where the lobe is expected to be) and the phenotypic rescue with presence of normal mushroom lobes in B. Size bars are 25 μm. (E-H) Head and eye phenotypes in wild type control (E) and in the ey^OK107/16 mutant in the presence of different UAS-Eyeless rescue constructs. Note that expression of full-length Eyeless completely rescues the eye and head phenotypes (F) whereas an Eyeless transgene encoding an Eyeless protein without the C-terminal domain (G) is not different from the siblings carrying the balancer chromosome (H) and yields flies with head and eye (arrowhead) defects and no phenotypic rescue. (I-L) Genotypes of the animals displayed in the corresponding panels on the same row (e.g. I represents the genotype of the animals shown in A and E).