(A, B) Single confocal sections through third larval instar wing discs, fixed and stained with α-Wg or α-Vg antibody, as indicated in panels. wg is activated by Notch signaling along the prospective anterior margin of the third instar disc, while vg is expressed in a Wg-dependent fashion in a broad zone straddling Wg expression along the prospective margin and sustains proliferation in the prospective wing blade (Couso et al., 1995; Rulifson and Blair, 1995; Kim et al., 1996; Klein and Arias, 1999; Micchelli et al., 1997). (C–E) Single confocal sections of wing discs as in (A, B), bearing (C) pygoS123, (D) ssdpL7 or (E) chipe55 mutant clones, stained with α-Vestigial (Vg) (red in merge); clones are marked by absence of GFP (green in merge); all discs were counterstained with DAPI (blue in merge), to label the nuclei (as internal control for the focal plane). Reduction of Vg expression within mutant clones of all three genes is best detectable in the ventral compartment (without Apterous expression) at a distance from the Wg source, as shown in high-magnification insets below panels (from marked squares, marked in merges; clones demarcated by white lines), as previously reported for dTCF mutant clones that are unable to respond to Wg signaling (Schweizer et al., 2003). (F–H) Single confocal sections of wing discs as in (C–E), bearing (F) ssdpL7, (G) chipe55 or (H) pygoS123 mutant clones, stained with α-Wg (red in merge); clones are marked by absence of GFP (green in merge), and discs were counterstained with DAPI; insets below panels are high-magnification views of selected clones (marked by squares in merges) revealing ectopic activation of Wg along the edge of the clone (signifying activation by Notch signaling derived from adjacent wt cells, combined with loss of Wg-mediated repression within the clone; Rulifson and Blair, 1995; Rulifson et al., 1996). Note also that, in the dorsal disc (the Apterous territory), chipe55 mutant clones barely survive (indicated by asterisks in the merge in G), compared to ssdpL7 mutant clones that appear to grow normally, like pygoS123 mutant clones; however, in the absence of Apterous, i.e. in the ventral compartment, chipe55 mutant clones grow normally (see clone marked by arrow in E). Collectively, our clonal analysis indicates that ChiLS is required for Wg and Notch responses in the prospective wing blade and margin, consistent with previous results (Morcillo et al., 1996; Morcillo et al., 1997; van Meyel et al., 2003) similarly to Pygo (Belenkaya et al., 2002; Parker et al., 2002; Thompson et al., 2002), although Chip exhibits a far more stringent requirement for proliferation in the Apterous territory than either SSDP or Pygo.