Fig. 7. Trl is not required for repression or activation of homeotic genes in imaginal disks. (A,B) Wing (W) and haltere (H) imaginal disks with Trl mutant clones, which are marked by the lack of GFP signal, stained with antibodies against GFP (green) and Ubx (red in A) or ßgal (red in B) protein. In all cases, the Minute technique was used and clones were analysed 96 hours after clone induction. (A) No misexpression of Ubx was observed in Trl mutant clones (left). Trl mutant clones induced in pho homozygotes (right) do not show anymore misexpression of Ubx than is seen in pho homozygotes alone (compare with Fig. 3A). (B) Trl mutant clones induced in transgenic larvae that express the PRE_1.6 (left) or MCP725 (right) reporter transgenes. Expression of both reporter transgenes is confined to the posterior half (ps 6; marked by asterisk) of the haltere disk in wild-type animals. No misexpression of the PRE_1.6 reporter gene is detected in Trl mutant clones; the MCP reporter gene shows patchy expression of ß-gal in 10-20% of the wing disks, independent of whether the cells are wild-type or mutant for Trl, but is not seen in this disk. (C) Requirement for Pho, but not GAGA-binding sites for silencing. (Top) sequence of the 567 bp PRE_D fragment containing five GAGAG sites (green) and six binding sites for Pho protein (red). All six Pho-binding sites or all five GAGAG motives were mutated to obtain PRE_DPhomut and PRE_DGAGAmut, respectively; base substitutions are indicated above the sequence. (Below) X-Gal staining of wing imaginal disks carrying the indicated reporter transgene. In PRE_D and PRE_DGAGAmut transformants, the transgene is silenced in wing disks. No silencing is observed in PRE_DPhomut, transformants (Fritsch et al., 1999). (D) Requirement for trx but not Trl in maintaining homeotic gene expression in imaginal disks. Haltere (H) and third leg (L) imaginal disks with Trl (top) or trx (bottom) mutant clones stained with antibodies against GFP (green) and Ubx (red). In both cases mutant clones are marked by the lack of GFP signal and clones were analysed 96 hours after clone induction, the Minute technique was only used in the case of Trl. Ubx expression is unaffected in Trl mutant clones (white arrowheads), whereas trx mutant clones show a complete loss of Ubx signal (empty arrowheads).

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