Involvement of winged eye encoding a chromatin-associated bromo-adjacent homology domain protein in disc specification

Abstract

How organ identity is determined is a fundamental question in developmental biology. In Drosophila, field-specific selector genes, such as eyeless (ey) for eyes and vestigial (vg) for wings, participate in the determination of imaginal disc-specific identity. We performed gain-of-function screening and identified a gene named winged eye (wge), which encodes a bromo-adjacent homology domain protein that localizes at specific sites on chromosomes in a bromo-adjacent homology domain-dependent manner. Overexpression of wge-induced ectopic wings with antero-posterior and dorso-ventral axes in the eye field in a region-specific Hox gene-(Antennapedia) independent manner. Overexpression of wge was sufficient for ectopic expression of vg in eye discs. A context-dependent requirement of wge was demonstrated for vg expression in wing discs and for expression of eyes absent (eya), a control gene for eye development downstream of ey, in eye discs. In contrast to vg, however, overexpression of wge inhibited EY-mediated expression of eya. Consistent with colocalization on polytene chromosomes of WGE and Posterior sex combs (PSC), a Polycomb group gene product, we demonstrated an antagonistic genetic interaction between wge and Psc. These findings suggest that wge functions in the determination of disc-specific identity, downstream of Hox genes.

Fig. 1.

Identification of winged eye, the overexpression of which induces ectopic wings in the eye field and its Antp-independent function on ectopic wing induction. (A) Schematic representation of the genomic regions of wge. The black boxes represent the wge ORF. The direction of GS vector-mediated transcription, the insertion site of the P element GS15923, and previously estimated transcript of CG31151 (RA) from EST clones are indicated. The broken line represents the deletion in wge⁴⁰. (B) WGE-mediated induction of ectopic wings in ey-GAL4, UAS-wge fly. Arrowhead indicates an ectopic wing. (C) Higher magnification of B. WGE-mediated ectopic wings have the costa with spine bristles (dotted lines), the triple row of bristles (bold line), the double row of bristles (narrow line), and the posterior rows of hairs (the remaining wing margin). (D) These structures are formed at the anterior-proximal part of wing (dotted line), anterior wing margin (bold line), distal wing margin (narrow line), and posterior wing margin in the wild-type wing, respectively. (E) GAL4-dependent induction of wge. RT-PCR was performed with hs-GAL4, GS15923 larvae (+), and GS15923 (-). rp49 was used as an internal control. (F) VG-mediated wing-like outgrowth without margin bristles formed in the eye field of the ey-GAL4, UAS-vg fly. Arrowhead indicates ectopic outgrowth. (G) Antp-independent induction of ectopic wings in the ey-GAL4, UAS-wge, UAS-Antp-IR fly. Arrowhead indicates ectopic wing with margin bristles. (H) Induction of ectopic wing in the ey-GAL4, UAS-N^act, UAS-Antp fly. Arrowhead indicates an ectopic wing with margin bristles. (I) Eye field of the ey-GAL4, UAS-N^act, UAS-Antp, UAS-Antp-IR fly.

Fig. 2.

WGE-mediated induction of VG and WG and WGE-mediated suppression of EYA in eye discs. (A and D–F) Immunostaining of eye discs of ey-GAL4, UAS-wge larvae with antibodies against ANTP (A), VG (green) and EYA (red purple) (D), WG (E), and β-galactosidase (F). (F) An eye disc of an ey-GAL4, UAS-wge, eya-lacZ larva. (C) X-gal staining (blue) of an eye disc of an ey-GAL4, UAS-wge, vgBE-lacZ, UAS-p35 larva. To avoid vgBE-lacZ-mediated cell death, p35, a cell death inhibitor (22), was coexpressed. (B, G–I) Immunostaining of eye discs of wild-type larvae with antibodies against ANTP (B), VG (green) and EYA (red purple) (G), WG (H), and β-galactosidase (I). (I) An eye disc of an eya-lacZ transgenic larva. Staining is merged with the bright field image (except C). In all images, posterior is to the right and dorsal is up.

Fig. 3.

A context-dependent requirement of wge for vg expression in wing discs and for eya expression in eye discs. (A) Lack of wge transcript in wge⁴⁰ mutant. RT-PCR was performed with first- and second-larval stage wild-type (WT) and wge⁴⁰. rp49 was used as an internal control. (B) Developmental defect of wge⁴⁰. Wild-type larvae (left side) and wge⁴⁰ larvae (right side) at the indicated times AED are represented. (Scale bars: 1 mm.) (C–F) wge mutant clones were introduced in wing discs (C and D) and eye discs (E and F) at 48 h (C and E) and 72 h AED (D and F). Immunostaining (red purple) with antibody against VG (C and D) and EYA (E and F) is merged with GFP (green). wge mutant clones lack GFP signals. Yellow arrowheads indicate cells with no VG (C) or EYA expression (E) within the clones. Blue arrows indicate cells expressing VG (C) or EYA (E) within the clones. In D, yellow arrowheads indicate wge mutant clones with no VG expression. In C and D, dorsal is to the left and anterior is up. In E and F, posterior is to the right and dorsal is up.

Fig. 4.

BAH domain-dependent localization of WGE and colocalization with PSC at specific sites of polytene chromosomes. (A and B) FLAG-tagged wild-type WGE and FLAG-tagged mutant protein (ΔBAH) lacking the BAH domain were expressed in a salivary gland (nonspecific expression of target genes are induced in a salivary gland by the GAL4/UAS system). The polytene chromosomes of ey-GAL4, UAS-FLAG-wge larvae (A) and ey-GAL4, UAS-FLAG-wgeΔBAH larvae (B) were stained with an anti-FLAG antibody. Immunostaining (red) and DAPI staining (DNA, blue) is merged. (C) The binding sites of WGE (red color is changed to white) were analyzed with DAPI staining (blue) in higher magnifications. Some WGE signals are overlapped with DAPI staining (circles), and others do not overlap with DAPI staining (arrowheads) in a merged picture. (D) Colocalization of PSC with WGE on polytene chromosomes. The polytene chromosomes of ey-GAL4, UAS-HA-wge larvae were stained with anti-HA antibody (red) and anti-PSC monoclonal antibody (green). Almost all PSC binding sites are coincident with some of WGE binding sites.