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Fig. 4. pygo is required for Wg signaling in the embryo. (A-E) Micrographs of cuticles of wild-type (A), wgCX4 (B), pygo10 maternal/zygotic mutant (C), a pygo10 maternal mutant without (D) or with (E) a P[UAS- pygo] transgene. The mothers in D,E are heterozygous for P[Da-Gal4]. The absence of maternal and zygotic pygo results in a denticle lawn similar to that observed in wg mutants (compare panel B with C). Paternal rescue of the pygo germline clone gives a variable phenotype (see Table 1 for more details), with most cuticles having four to seven abdominal denticles (the one in D has six). Further rescue was observed with the P[UAS- pygo] transgene driven by P[Da-Gal4] (Table 1), with the example shown in E having seven denticles (arrow indicates the partial fusion of the fourth and fifth abdominal denticles belts). (F-N) Confocal images of wild type (F,I,L), wgCX4 (G,J,M) or pygo10 maternal/zygotic mutant embryos (H,K,N) stained with antibodies against En (F-H), Wg (I-K) or Eve (L-N). The embryos in F-K are at mid-stage 10 and the ones in L-N are at stage 13. Embryos that lack zygotic pygo were unambiguously identified by the absence of eve-lacZ staining. Note that at mid-stage 10 (judged by the extent of stomodeum invagination), when wild-type and wg mutants are at full germband extension (most clear in F and I), the pygo mutants have incomplete extension (clear in H but even more obvious in K). As described in the text, the En and Wg stripes are largely absent at this stage in pygo mutants (H,K), though the residual epidermal En expression (arrows) is not observed in wg mutants (G). The Eve pericardial expression (arrow in L) is absent in wg and pygo mutants (arrows in M and N), while Eve expression in the CNS is present (arrowheads).
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-cop. The location and orientation of the EP(3)1076 transposon is also shown. Besides the Gal4-dependent dominant phenotypes seen with EP(3)1076, the transposon also causes a Gal4-independent recessive phenotype (referred to as pygoEP). (B) Depiction of two additional pygo loss-of-function alleles created by imprecise excision of EP(3)1076. pygo10 contains half of the transposon and removes the splice acceptor site in the 5' UTR intron of the pygo gene, and the first 295 residues of the Pygo ORF. It does not affect rod (see Results). The pygo9 mutation extends further upstream of pygo, and inactivates rod as well. In situ hybridization of embryos (C-F) and wing imaginal discs (G,H) with a probe for pygo. Preblastoderm wild-type embryo (C) shows high levels of staining that are absent in pygo10 germline clones (D). At stage 10, wild-type embryos (E) show ubiquitous staining at a much lower level than in C (the preparation was allowed to develop much longer). Stage 10 embryos maternally and zygotically mutant for the pygo10 allele (F; identified by their altered morphology) presumably indicate the level of background staining under these conditions. Late third instar wing imaginal discs show low levels of ubiquitous signal (G), while discs containing random clones of Gal4-expressing cells (via an actin promoter) in a EP(3)1076 background show patches of cells (arrows) with much higher levels of pygo transcripts (H).
