Fig. 1. Ubiquitously expressed Engrailed requires exd and hth to repress slp in vivo. Repression of slp by En is abrogated by loss of either hth or exd function. Embryos were collected for 1 hour, aged for 3.5 hours and heat shocked at 37°C for 10 minutes to induce En expression in hs-En embryos, which carry a hs-inducible transgene expressing full-length En). They were then aged for an additional 15 minutes, fixed and stained for slp RNA (dark blue, by in situ hybridization) and for ß-galactosidase protein (A,B,D,E) (orange, anti-horseradish-peroxidase stain). lacZ is expressed from a ftz-lacZ transgene on the TM3 balancer chromosome. (A) Wild type (no hs-En, genotype TM3/hth^P2 or TM3/TM3, which are indistinguishable). (B) hs-En, TM3 containing (TM3 carries a wild-type hth allele); notice the almost complete repression of slp by hs-En. (C) Control to show that TM3 itself does not affect repression of slp by hs-En (genotype hs-En; +/+, stained in parallel with the others). (D) An hth mutant (genotype hth^P2/hth^P2, distinguishable in the same population as in A by the absence of ß-galactosidase staining). (E) An hth mutant with hs-En (genotype hs-En; hth^P2/hth^P2, inferred from the absence of ß-galactosidase staining; this and the embryo in B were stained together in the same tube). Notice the weak repression of slp relative to B. (F-H) Embryos in F and H, designated `mat.exd<sup>-</sup>' below (`m-' in the figure), were derived from germline clones in the mother that were homozygous for the null exd allele exd¹; therefore, no maternally derived Exd was present. These females with exd^- germlines were crossed either to wild-type males, to generate the embryo in F, or to males homozygous for a hs-En transgene, to generate the embryo in H. Wild-type females were crossed to the hs-En males to generate the embryo in G. (F) mat.exd^-, without hs-En. (G) exd⁺, with hs-En. (H) mat.exd^-, with hs-En. Notice the increase in slp RNA when Exd is missing (H versus G), indicating a requirement for Exd in order for En to efficiently repress slp. These embryos were also stained for the female-specific protein Sex-lethal, to indicate whether they were male or female. Because exd is on the X chromosome, females received a wild-type copy of the exd locus from their fathers, whereas males did not. The embryos shown were male, and so they received their only exd allele from their mother (`z-' or `z+'). A paternal-only contribution to exd function (that is, in females derived from females with exd^- germlines) significantly increased the ability of hs-En to repress slp (data not shown).