<p>(A) Posterior spiracle (PS) of a 1<sup>st</sup> instar wild-type <i>Drosophila</i> larva. The filzkörper is highlighted by red asterisks. (B, C) <i>rpr</i> mRNA (green) expression in stage 11 wild-type (B) and <i>ct</i> mutant (C) embryos. Spalt (Sal) protein (blue) labels stigmatophore precursor cells, Cut (Ct) protein (red, nuclear) marks spiracular chamber and filzkörper precursor cells and the apical membrane marker Crb (red) outlines the cells. Small, green arrows in (C) mark <i>rpr</i> positive spiracular chamber and filzkörper precursor cells in the eighth abdominal segment (A8) of <i>ct</i> mutant embryos. (D, E) Over-expression of the apoptosis sensor UAS-<i>Apoliner</i> using the <i>arm</i>-GAL4 driver in stage 11 wild-type (D) and <i>ct</i> mutant (E) embryos. Small, green arrows in (E) mark apoptotic cells in PS precursor cells (A8) of <i>ct</i> mutant embryos. (F, G) TUNEL stainings in wild-type (F) and <i>ct</i> mutant (G) embryos. Closed arrowhead in (G) marks TUNEL-positive cells in <i>ct</i> mutants, which are absent in wild-type embryos (F). (H, I) Co-localization of GFP protein and <i>rpr</i> mRNA (H) or Cut protein (I) in stage 15 <i>rpr</i>-HRE-571 embryos. White circles mark the PS primordium. (J) Top: conservation blot of <i>rpr</i>-HRE-571 genomic region obtained from the UCSC genome browser (<a href="http://genome.ucsc.edu/" target="_blank">http://genome.ucsc.edu/</a>). Species used for generating blot are also shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002582#pgen.1002582.s002" target="_blank">Figure S2A</a>. Bottom: diagram of the <i>rpr</i>-HRE-571 deletion constructs tested. (K) EMSA using S2 sub-fragment with Ct binding sites either in wild-type (wt probe) or mutated (mut. probe) version and no protein (−), purified MBP protein (M), and purified Cut-MBP fusion protein consisting of the Cut repeat 3 and the Cut homeodomain (C). The black arrowheads indicate the specific DNA-protein complexes. Loading of equal amounts of labeled wild-type and mutated oligonucleotides is illustrated by formation of comparable amounts of unspecific DNA-protein complexes (black arrow). (L–O) Reporter gene expression in the PS of stage 15 embryos driven by the fragments described above. In the S2-Ctbs-GFP, line Ct binding sites within the <i>rpr</i>-HRE-571-S2 fragment are mutated. Spalt (Sal) and Cut (Ct) proteins label stigmatophore (blue) or spiracular chamber and filzkörper cells (red). Closed, yellow arrowheads in (N) and (M) mark reporter gene expression in filzkörper cells, whereas open, yellow arrowheads in (L) and (M) mark missing GFP expression.</p

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