Fig. 3. Essential role for pho and phol in the repression of homeotic genes in embryos and imaginal disks. (A) Wing imaginal disks from larvae stained with Ubx or Abd-B antibody (red in both cases). No Ubx or Abd-B expression is detected in wing imaginal disks of wild-type larvae or in phol homozygotes. In pho homozygotes, Ubx is misexpressed in some cells in the wing pouch; but Abd-B is not misexpressed. Strong misexpression of Ubx and Abd-B is observed in phol, pho double homozygous disks; misexpression of Ubx is confined to the wing pouch, the lack of misexpression in more peripheral parts of the wing disk is possibly the result of downregulation by Abd-B protein, which is expressed at higher levels in these regions. (B) phol function is required throughout development. Wing (W) and haltere (H) imaginal disks with clones of phol mutant cells, marked by the lack of GFP signal (green), were induced in pho homozygous larvae and disks were analysed 96 hours after clone induction by staining with antibody against Ubx (red). The Minute technique was used in this experiment to generate M+ phol—/M+ phol- cells that carry two copies of a wild-type Minute allele; this gives them a growth advantage relative to their M+ phol-/M- phol+ neighbours. Strong misexpression of Ubx is observed in clones in the wing pouch (filled arrowhead). The lack of misexpression in clones in peripheral regions of the wing disk and the reduction of Ubx signal in clones in peripheral regions of the haltere disk (empty arrowhead) might be due to downregulation by Abd-B. (C) Requirement for pho and Pho-binding sites for silencing in embryos. (Top) Embryos carrying the PRE_D reporter gene (Fritsch et al., 1999) show ß-gal expression restricted to parasegments 6-12 (ps 6-12) in late (stage 13) embryos. In PRE_DPhomut transformant stage 13 embryos, repression anterior to ps 6 is lost and the reporter gene is active in all segments because of mutation of the six Pho-binding sites in PRE_D. (Below) Expression of the BP01 reporter gene is restricted to ps 6-12 in wild-type (wt) stage 16 embryos (Müller and Bienz, 1991). pho mutant stage 16 embryos show misexpression of the reporter gene in the nervous system (asterisks) and in the epidermis. In all cases, embryos were stained with anti-ß-gal antibodies and are oriented anterior leftwards, dorsal upwards; the anterior boundary of ps 6 is indicated by an arrowhead.