Volume 3, Issue 3, Pages 297-307 (March 1999) twin of eyeless, a Second Pax-6 Gene of Drosophila, Acts Upstream of eyeless in the Control of Eye Development  Thomas Czerny, Georg Halder, Urs Kloter, Abdallah Souabni, Walter J Gehring, Meinrad Busslinger  Molecular Cell  Volume 3, Issue 3, Pages 297-307 (March 1999) DOI: 10.1016/S1097-2765(00)80457-8

Figure 1 Conservation of the Toy Sequence within the Pax-6 Protein Family (A) The amino acid sequence of Toy is compared with Ey (Quiring et al. 1994) and Pax-6 proteins from human (h; Ton et al. 1991), sea urchin (su; Czerny and Busslinger 1995), and C. elegans (Vab-3; Chisholm and Horvitz 1995). Sequences that could not be unequivocally aligned are abbreviated by dotted lines. Identical amino acids are highlighted by gray overlay. Arrowheads and asterisks indicate known and toy/ey-specific intron positions, respectively. Arrows denote the two peptides used to design oligonucleotides for PCR amplification of toy paired domain cDNA. Numbers refer to the protein length. (B) Pax-6 gene duplication during insect evolution. DNA sequences coding for the N- and C-terminal part of the Pax-6 paired domain were amplified from genomic DNA of the indicated species by two separate PCR reactions. Numbers indicate the occurrence of PCR clones with identical Pax-6 sequences. Molecular Cell 1999 3, 297-307DOI: (10.1016/S1097-2765(00)80457-8)

Figure 2 Comparison of toy and ey Expression during Drosophila Development Transcripts of toy (left) and ey (right) were detected by whole-mount in situ hybridization. All embryos are oriented with anterior to the left and are shown as lateral (A, B, and D–H) or dorsal (C and I–M) views. (A–C) Cellular blastoderm (stage 5). (D) Early gastrula (stage 6). (E and F) Germband extension (stage 10). (G and H) Germband retraction (stage 13). (I–M) Embryos after head involution (stage 16). Optical sections through the dorsal (I and K) and ventral (L and M) region of the same embryos are shown. (N and O) Eye-antennal imaginal discs of crawling third instar larvae. Arrowheads indicate the dorsal midline (C), optic lobe (G, H, I, and K), and morphogenetic furrow (N and O). An arrow points to the primordia of the eye imaginal discs (I and K). a, antennal disc; e, eye disc. Molecular Cell 1999 3, 297-307DOI: (10.1016/S1097-2765(00)80457-8)

Figure 3 Activation of the Eye Developmental Pathway by Toy (A) Ectopic eye formation by targeted expression of Toy in leg imaginal discs. Transgenic flies carrying a toy minigene under the control of GAL4 upstream activating sequences (UAS-toy) were crossed with the transgenic line dppdisk-GAL4 (Staehling-Hampton et al. 1994). Targeted misexpression of Toy induced the formation of extra eyes on legs (arrows) and wings with 100% efficiency and on the antennae with 30% penetrance. (B) Induction of ey transcription by ectopic Toy expression. ey mRNA was detected by whole-mount in situ hybridization in leg imaginal discs of third instar larvae, which were homozygous for the so1 mutation and contained the dpp-GAL4 and UAS-toy transgenes. (C) toy transcription is not induced in leg imaginal discs of so1 mutant larvae, which ectopically express a UAS-ey transgene. (D) ey but not toy (E) transcription is activated by ectopic expression of mouse Pax-6 in leg imaginal discs of larvae carrying dpp-GAL4 and UAS-mPax6 transgenes. Molecular Cell 1999 3, 297-307DOI: (10.1016/S1097-2765(00)80457-8)

Figure 4 Toy Acts Upstream of ey in the Eye Developmental Pathway (A and B) Normal expression of toy in ey2 and eyR mutant embryos at stage 16. Arrows point to toy expression (detected by whole-mount in situ hybridization) in the primordia of the eye imaginal discs. (C) Normal leg of an ey2 mutant fly. (D) Ectopic eye induced by dpp-GAL4-driven Toy expression on the leg of a fly containing a wild-type (wt) ey gene. (E) dpp-GAL4-driven Toy expression is unable to induce the formation of ectopic eyes in an ey2 mutant background. (F) Complete absence of the compound eye in the same ey2 mutant fly whose leg is shown in (E). Molecular Cell 1999 3, 297-307DOI: (10.1016/S1097-2765(00)80457-8)

Figure 5 Identification of Pax-6 Binding Sites in the Eye-Specific Enhancer of ey (A) DNA sequences of the second ey intron surrounding the transposon insertions in eyR and ey2 mutants. The three Pax-6 binding sites A, B, and C (boxed) are shown together with the extent of the Pax-6 DNase I footprints (brackets) and the specific mutations introduced. Numbers refer to nucleotide positions in the ey intron (Hauck et al. 1999). (B) DNase I footprint. A 3′ end-labeled DNA fragment containing the ey enhancer was incubated with mouse Pax-6 protein (30 ng–3 μg) followed by DNase I digestion and gel electrophoretic analysis. The same probe cleaved at purines was used as a DNA sequence ladder (G+A). The analysis of naked DNA is shown in lane (−). Pax-6 (1 μg; lane +) was unable to bind to the mutated (mut) intron probe. Molecular Cell 1999 3, 297-307DOI: (10.1016/S1097-2765(00)80457-8)

Figure 6 Distinct DNA-Binding Properties of Toy and Ey Due to a Single Amino Acid Substitution (A) Alignment of the Pax-6 binding sites A, B, and C of the ey enhancer with the high-affinity binding site CD19-2 (A-ins) (Czerny et al. 1993) and the consensus recognition sequence of the Pax-6 paired domain (Czerny and Busslinger 1995). (B) Structure of the N-terminal part of the paired domain. A schematic diagram of the paired domain structure (Xu et al. 1995) indicates the positions of β sheets (β1 and β2), β turns (τ1, τ2), and the first α helix (α1) within the N-terminal part of the paired domain. (C) Differential binding of Toy and Ey to the ey enhancer. Equimolar amounts of in vitro translated Toy, Ey, and mPax-6 proteins were analyzed by EMSA for binding to the ey A, B, C, and CD19-2(A-ins) sites. Quantitation of the protein–DNA complexes by phosphorimager analysis revealed a similar DNA-binding affinity for mouse Pax-6 and Toy, while the binding activity of Ey was 12-fold lower than that of Toy for all four recognition sequences. (D) Increased DNA-binding activity of Ey due to a single amino acid substitution. EMSA of the indicated in vitro translated proteins demonstrated a 10-fold increase in DNA-binding affinity of EyN due to the G50N mutation. Molecular Cell 1999 3, 297-307DOI: (10.1016/S1097-2765(00)80457-8)

Figure 7 The Toy-Binding Sites Are Essential for Activation of the Eye-Specific Enhancer of ey in the Embryo (A and B) Expression of the wild-type (wt) or mutant (mut) E0.37-lacZ transgene in stage 16 embryos. Mutation of the Pax-6 binding sites in the ey enhancer (Figure 5A) almost completely abolished transgene expression in the eye anlagen (B) where β-galactosidase staining was readily detected in embryos carrying the wild-type transgene (arrows in [A]). (C) Expression of the E0.37-lacZ transgene in the eye imaginal disc (e) of third instar larvae. Reporter gene expression (detected by X-Gal staining) is strongest just anterior to, within, and posterior to the morphogenetic furrow (arrowhead). (D) Mutation of the Pax-6 binding sites in the ey enhancer did not affect transgene expression in the eye imaginal disc. (E and F) Feedback regulation of ey, but not toy, by So, Eya, and Dac. dpp-GAL4-driven coexpression of So, Eya, and Dac in the antennal disc (a) induces expression of the endogenous ey (E, arrowhead) but not toy (F) gene, as detected by whole-mount in situ hybridization. Molecular Cell 1999 3, 297-307DOI: (10.1016/S1097-2765(00)80457-8)

Figure 8 Regulatory Relationships between Genes Involved in Drosophila Eye Specification The network of regulatory genes specifying eye development is established in a biphasic manner. A linear pathway (red arrows) is responsible for initiating expression of ey, so, eya, and dac in the developing eye disc, while feedback control (green arrows) contributes to their regulation during larval development. See text for further explanation. Molecular Cell 1999 3, 297-307DOI: (10.1016/S1097-2765(00)80457-8)