Notch signaling induces eyg transcription in eye disc. (A) eyg^M3-12/TM3 fly is an enhancer trap line with a P[lacW] inserted in the eyg locus. The mini-white reporter in P[lacW] reflected the eyg expression pattern and in eyg^M3-12/TM3 shows the eye pigmentation pattern in an anterior-central region of adult eye. (B) The reporter expression in eyg^M3-12/TM3 is reduced in the N^ts mutant background. The eggs were laid at 18°C, raised at 18°C for 5 days, then shifted to 29°C until adults eclosed. The eye size is reduced. (C-F) Clones of ectopic N^act (marked by GFP, green) caused tissue overgrowth in eye-antennal disc. (C) DIC image. (D,F) eyg-lacZ (eyg^M3-12, stained by anti-β-galactosidase, red) expression is activated (arrow) within the N^act clones in eye discs except in the wg domain (arrowhead). (E) WG (stained by anti-WG, blue) is enhanced (arrowhead) within the wg endogenous expressing domain. (G-L) In most Su(H)^SF8 clones (marked by the absence of GFP, green) eyg-lacZ (eyg^M3-12, stained by anti-β-galactosidase, red) expression were not detected. But in a few clones, the mutant cells at the border of the clone still expressed eyg-lacZ.

eyg is induced at new DV border created by Ser- or Dl-expressing clones. (A-C) In Dl^rev10, Ser^RX82 and eyg^M3-12 triple heterozygous mutant, the eye size and eyg expression (eye pigmentation based on the mini-white reporter in eyg^M3-12) were both reduced (1% of flies are like A; 98% are like B; 1% are like C). (D-I) Ectopic expressing clones were marked by GFP (green). Discs were stained using anti-Elav (blue) and anti-β-galactosidase (eyg^M3-12, red) antibodies. (D-F) When the Ser-expressing clone is located in the dorsal side near the DV midline of eye disc, eyg-lacZ is induced non-autonomously at the border of the clone (arrow). E,F are higher magnifications of D. (G-I) When the Dl-expressing clone is located in the ventral side near the DV midline of eye disc, eyg-lacZ is induced non-autonomously at the border of the clone (arrow). H,I are higher magnifications of G.

eyg mediates the growth promoting function of Notch signaling. eyg¹/eyg^M3-12 mutants are eyeless with complete penetrance (see Fig. 1D). (A) In eyg¹, H^E31/eyg^M3-12 the eye size is partially restored. The eye size is also partially restored in (B) dpp-GAL4 UAS-N^act (abbreviated as dpP>N^act) and (C) dpp>Su(H) in the eyg¹/eyg^M3-12 mutant background. (D,E) When one copy of H was removed in eyg^M3-12, H^E31/eyg^M3-12, the flies have complete head structures but no eye (D) and eyg-lacZ (anti-β-galactosidase, red) is detected in antennal discs, but not in eye discs (E). The disc is also stained with anti-DAC (green) to mark the antennal disc. (F) In ey>N^DN flies, the eye formation is completely inhibited. (G) When eyg and N^DN were co-expressed (ey>N^DN+eyg), the eye size was partially restored. (H) In ey>H flies, the eye formation is completely abolished. (I) In ey>H+eyg flies, the eye size is partially restored.

Effect of N induction in eyg null mutant. N^act was induced by ey-GAL4 (abbreviated as ey> N^act) in eyg^M3-12 homozygous mutant background. (A) Eighty percent of the pharates have head with no eyes. (B) Twenty percent have partially restored eye. (C) A few flies have a duplicated antennae. (D-F) The antennal discs are restored in size and have eyg-lacZ expression (green). (F) A disc has triplicated antennal fields (indicated by the eyg-lacZ expression). Occasionally one of the antennal fields can be recognized morphologically but lacks eyg-lacZ expression (not shown). (D-F) The endogenous eye field (based on the location of the optic stalk and Bolwig nerve; arrow) is highly reduced, lacks eyg-lacZ expression and has no photoreceptor differentiation (ELAV-staining, red). (D,E) In about 36% of the clones, an extra eye field (arrowhead) is induced dorsal to the endogenous eye field. The extra eye field can have eyg-lacZ (green) expression and photoreceptor differentiation (ELAV, red). The ocellus is marked by ELAV (^*). (G) The eyg-lacZ expression pattern (green) in wild type is used for comparison.

Notch induces upd expression. (A) upd-lacZ (anti-β-galactosidase, red) is expressed at the junction of optic stalk and the posterior margin of eye disc. (B,C) upd-lacZ (red) is induced (arrowhead) in the N^act-expressing clones (marked by GFP, green), but only when the clone is located at the posterior margin of the eye disc. Even large clones in the central region of the eye disc and in antenna disc caused no induction on upd-lacZ. (D,E) N^DN-expressing clone (marked by GFP, green) inhibited upd-lacZ (red) expression (arrow). Whereas ey>N^DN caused the complete absence of eye development (see Fig. 4F), ey>N^DN+upd (F) completely restored the eye size. (G,H) upd RNA in situ hybridization. (G) upd is expressed at the center of the posterior margin in wild-type late second instar eye-antenna disc. (H) N^ts eggs were laid at 18°C for 24 hours, cultured at 18°C for 6 days, then shifted to 29°C for 24 hours and dissected for hybridization. Only males were selected for disc dissection. The N^ts discs can be further identified by the reduction of eye disc size.