SO induces ectopic eyes in the antennal disc. (A–C) Scanning electron micrographs of adult normal and ectopic eyes. (D–S) Confocal images of 3rd instar eye-antennal imaginal discs. (A) Wild type. (B) cb41-GAL4/UAS-so, the yellow arrow marks the ectopic eye. (C) cb49-GAL4/UAS-optix, the yellow arrow marks the ectopic eye on the ventral portion of the head. (D) cb41-GAL4/UAS-GFP, the GAL4 line drives expression within the developing eye, ocelli and a large portion of the antenna. (E) cb41-GAL4/UAS-so, ELAVexpression marks the ectopic eye within the antennal disc. (F) cb41-GAL4/UAS-so, ELAVand EYA are distributed in subsets of cells within the cb41 expression pattern. (G) cb41-GAL4/UAS-so, DAC is also distributed in a smaller subdomain of the cb41 expression pattern. (H) cb41-GAL4/UAS-ey, expression of ey has a minimal effect on EYA distribution and no ectopic eyes are specified. (I) cb41-GAL4/UAS-ey, dac expression is not initiated by the expression of ey in this expression domain. (J–K) cb41-GAL4/UAS-toy, expression of toy is insufficient to induce either eya, dac or elav expression. (L) dpp-GAL4/UAS-GFP, GFP is distributed along the posterior-lateral margins of the eye disc and in a sector of the ventral antennal disc. (M) dpp-GAL4/UAS-ey, expression of ey is sufficient to induce ectopic eyes. (N–O) dpp-GAL4/UAS-so, expression of so induces the expression of eya, dac and induces eye formation in the antenna. (P) cb49-GAL4/UAS-GFP, expression of the GAL4 line is restricted to the distal most regions of the antennal disc. (Q–S) cb49-GAL4/UAS-so, note the ectopic photoreceptors are located at a distance from the cells that express GAL4. Genotypes are listed at the top right of each panel. Visualized molecules are in the top right of each panel. Anterior is to the right.

SO and OPTIX induce ectopic eyes in different cell types. (A–(D) Confocal images of 3rd instar eye-antennal imaginal discs. (E–G) Light microscope images of adult flies. (A) cb41-GAL4/UAS-GFP, note that the GAL4 line is expressed widely within the antennal disc. (B) cb41-GAL4/UAS-so, note the ELAV-positive cells lie within the broader GAL4 expression pattern. (C) cb309-GAL4/UAS-GFP, note the GAL4 expression pattern is in the medial–proximal segments of the antennal disc. (D) cb309-GAL4/UAS-optix, note that the ELAV-positive cells are located in regions of the antennal disc that are distinct from those that are transformed by so. (E) Wild type. (F) rn-GAL4/UAS-so, note that expression of so within the wing causes the wing to shrink in size and has patterning defects. (G) rn-GAL4/UAS-optix, note the replacement of wing tissue with retinal tissue. Genotypes are listed at the bottom right of each panel. Visualized molecules are in the top right of each panel. Anterior is to the right.

Schematic diagrams of SO deletion and SO/OPTIX chimeric proteins along with summary of rescue and overexpression assays. Individual domains of SO and OPTIX proteins were determined from published reports. All deletions and chimeric proteins were used in ectopic eye experiments (Fig. 4), so mutant rescue experiments (Figs. 5 and 6) and overexpression experiments (Fig. 7). Results from these figures are shown in tabular form.

Induction of ectopic eyes by SO/OPTIX chimeric proteins. (A–C) Light microscope images of whole adult flies. (A) cb41-GAL4/UAS-so/optix NT chimera, the arrow marks the location of the ectopic eye. The ectopic eye has partially merged with the normal compound eye. (B) cb41-GAL4/UAS-so/optix CT chimera, arrow marks the location of the ectopic eye. Note that the location is slightly different than that seen in panel A. (C) cb41-GAL4/UAS-so/optix NT CT chimera, the arrow marks the location of the ectopic eye. Note that in this case the ectopic eyes are very small in size compared with those depicted in panels A and B. Expressed proteins are listed at the top left of each panel. Anterior is to the right.

Rescue of so mutants by SO deletion and SO/OPTIX chimeric proteins. (A–O) Scanning electron micrograph of adult compound eyes and head. (A) Wild type. (B) so¹, note the complete absence of the compound eyes. (C) so¹; ey-GAL4/UAS-so, the compound eye is restored to near wild-type levels. (D) so¹; ey-GAL4/UAS-optix, expression of optix is insufficient to rescue the so¹ mutant. (E) so¹; ey-GAL4/UAS-so ΔNT, rescue is similar to wild-type so. (F) so¹; ey-GAL4/UAS-so/optix NT chimera, rescue is similar to wild type. (G) so¹; ey-GAL4/UAS-so ΔSD, no rescue. (H) so¹; ey-GAL4/UAS-so/optix SD chimera, no rescue. (I) so¹; ey-GAL4/UAS-so ΔHD, no rescue. (J) so¹; ey-GAL4/UAS-so/optix HD chimera, compound eye development is partially rescued. (K) so¹; ey-GAL4/UAS-so ΔCT, rescue is similar to wild type. (L) so¹; ey-GAL4/UAS-so/optix CT chimera, no rescue. (M) so¹; ey-GAL4/UAS-so ΔNT CT, rescue is similar to wild type. (N) so¹; ey-GAL4/UAS-so/optix NT CT chimera, no rescue. (O) so¹; ey-GAL4/UAS-so/optix SD HD chimera, no rescue. Anterior is to the right.

Rescue of so^D by SO deletion and SO/OPTIX chimeric proteins. (A–F) Scanning electron micrographs of adult compound eyes and heads. (A) so^D/+, note that heterozygotes completely lack the compound eyes. (B) so^D/+; ey-GAL4/UAS-so, note that expression of wild type so partially restores eye development. Multiple copies of the UAS-so insertion increase the number of ommatidia (data not shown). (C) so^D/+; ey-GAL4/UAS-optix, no rescue. (D) so^D/+; ey-GAL4/UAS-so ΔNT, note that the eye is partially restored. (E) so^D/+; ey-GAL4/UAS-so/optix NT chimera, note that the compound eye is partially restored. (F) so^D/+; ey-GAL4/UAS-so/optix HD chimera, note that the compound eye is partially restored. The remaining deletion and chimeric proteins listed in Fig. 3 did not rescue the no-eye phenotype of so^D. All genotypes are listed at the bottom left of each panel. Anterior is to the right.

The C-terminal tail of OPTIX inhibits eye development. (A–F) Scanning electron micrographs of adult compound eyes. (G) Light microscope image of an adult retinal section. (A) GMR-GAL4/UAS-optix, note that the eye is devoid of ommatidia. The eye field has a glassy appearance and is replete with small bristles. (B) GMR-GAL4/UAS-so/optix NT chimera, note that the eye is slightly rough. The posterior edge of the eye has the severest roughening. (C) GMR-GAL4/UAS-so/optix SD chimera, note that the eye is wild type in its appearance. (D) GMR-GAL4/UAS-so/optix HD chimera, note that the eye is wild type in appearance. (E) GMR-GAL4/UAS-so/optix CT chimera, note that the eye has a similar phenotype to that seen when wild-type optix is expressed (A). (F) GMR-GAL4/UAS-so/optix NT CT chimera, note that the eye has a similar phenotype to that seen when wild-type optix is expressed (A). Please note that the 3 instances in which the eye is severely altered (panels A, E, F) are the result of the expression of molecules that contain the C-terminal regions of the OPTIX protein. (G) GMR-GAL4/UAS-optix, note that there are no photoreceptor cells within this retinal section. Instead the eye is filled with pigment and bristle cells. Retinal sections of the eyes shown in panels E and F look identical to that shown here (data not shown).

Models for SO and OPTIX activity and regulation during eye development in Drosophila. (A) A portion of the eye specification cascade is shown. Our results suggest that in certain areas of the antenna expression of so but not toy or ey is sufficient to induce ectopic eyes (despite TOY and EY proteins binding to the promoter of so). One possible model is that there are additional (and yet to be identified) players that reside genetically between the two Pax6 genes and so. (B) We observed (in limited circumstances) that expression of so could non-autonomously induce ectopic eyes. A similar effect has been documented in the eye disc when dpp is overexpressed ahead of the morphogenetic furrow. Dpp is expressed in the antennal disc. One potential model is that SO interacts with or regulates Dpp, which in turn can induce ectopic eyes non-autonomously. (C) Our results suggest that the C-terminal tail plays a role in eye development and our sequence analysis has indicated that there are regions of amino acid conservation. In one model these regions of OPTIX might be bound to additional co-factors and that this helps to modulate OPTIX activity. (D) A schematic diagram showing an alignment of the C-terminal regions of OPTIX, SIX3 and SIX6. The red blocks are regions of the highest conservation that might serve as potential protein–protein interaction domains.