Expression array and DamID analyses. (A) Cluster analysis of expression arrays depicted as columns representing pairwise comparisons of transcript levels of da>Ci^Act, ptc^B98/ptc^6C, ci⁹⁴/ci⁹⁴, smo^Q/smo^Q and hh^AC/hh^13C to controls. Each lane represents one array experiment. Genes upregulated under normal or elevated Hh signaling are represented in rows as shades of green. bap, rdx, wg and en were previously shown to respond to Hh signaling in the embryo; hh, ato, CycE, drm and h have been shown to respond to Hh signaling in non-embryonic tissues. sna and ImpL2 are characterized in this study. (B) Above: Venn diagram indicating the number of genes linked to DamCi^Act and DamCi^Rep sites or both. Below: Overlap between expression array targets and genes linked to DamID sites.

Hh signaling in Drosophila embryonic visual primordia. (A-N) Lips of the optic lobe (OL) placode are outlined with dotted lines. Anterior is to the left, dorsal is at the top. Patterns of hh expression in the OL change in early (A), mid (B) and late (C) stage 11 embryos. Fas2 expression (D; red) marks the posterior optic lobe (POL) and is coincident with hh-lacZ at mid-stage (E; green). Fas2 staining (F; red) marks POL in hh^AC (hh^–). Full-length Ci (G; red) in the AOL and in Fas2-containing POL (H; green). (I) smo expression in AOL and POL. (J) Ptc protein (arrow) in AOL. (K) En (red) is absent from the OL but is present elsewhere where hh is expressed (arrow). (L) rdx expression in POL, in adjacent AOL and in cells dorsal to the OL (black arrow) and in ectodermal stripes (white arrows). (M) rdx expression in hh^AC (hh^–) is largely absent from the OL but is present in more dorsal cells (arrow). (N) rdx expression in en^l11/en⁷ is absent from ectodermal stripes but unaffected in the OL. (O) Diagram of Hh target gene activation in the optic primordium. Left: Fas2 (green) in the POL and Ptc protein in the adjacent AOL (brown). Middle: Ci^Act (red) in both the POL and adjacent AOL and Hh (blue) in the POL. Right: rdx (purple) in the POL and adjacent AOL.

ImpL2 is a Hh target in the tracheal pit of Drosophila. (A-P) Lateral views of stage 11 embryos. Anterior is to the left. hh (A) is expressed in a stripe of cells immediately anterior to each tracheal pit (dashed circle; pit identified by gap). Pit cells express ci RNA (B), stabilize Ci^FL (C,D) and express rdx (D). trh is expressed in a broad region that includes the pit (E) and trh expression in the pit is not diminished in hh^AC mutants (F). ImpL2 is non-uniformly expressed in the pit with highest levels in the most dorsal anterior cells (G). ImpL2 expression in the pit is severely reduced in hh^AC (H) and trh¹/trh² (I; arrow). Anti-GFP in a btl>GFP embryo reveals btl expression (J). ImpL2 expression in the pit expands similarly in btl>Ci^Act (K) and da>Ci^Act (L). ImpL2 expression in ectodermal cells dorsal to the pit (M; arrow) present in trh mutants (N; arrow) and btl>Ci^Act (O; arrow), and elevated in da>Ci^Act (P; arrow). (Q) Statistically significant DamCi^Act signal (orange bars) and Ci binding motif clusters (red boxes) in first intron and downstream of ImpL2; binding motif for Trh (green line) in first intron. Ci motif clusters match the motif YGSGDGGNC and are listed in Table S2 in the supplementary material. (R) Summary diagram of Hh target gene activation in the cells of the tracheal pit. Expression of hh (brown), ptc (blue), Ci (red), btl (gray), trh (light green) and Impl2 (dark green) in wild type (WT) and btl>Ci^Act mutants reveals the location of the tracheal pit relative to the AP compartment border (the interface of ptc and hh expressing cells), the extent of the tracheal primordium (defined by domains of btl and trh expression) and expansion of Impl2 to the entire tracheal primordium in the presence of ubiquitous Ci^Act.

Ci binds at known targets of Hedgehog signaling in Drosophila. (A-F) Averaged log2-transformed ratios of DamCi^Act/Dam alone (orange vertical bars) and DamCi^Rep/Dam alone (blue vertical bars) at statistically relevant regions of the genome (x-axes) for patched (ptc; A), wingless (wg; B), roadkill (rdx; C), atonal (ato; D), hedgehog (hh; E) and engrailed/invected (en/inv; F) genes, all of which had a significant DamID signal. Transcription units are indicated below. Red boxes indicate clusters of Ci binding motifs with at least two motifs matching the consensus YGSGDGGNC within a 600 bp window (for coordinates and sequence of the motifs see Table S1 in the supplementary material). Green boxes (A,D) indicate known enhancers (Forbes et al., 1993; Sun et al., 1998).

Genes in the odd skipped cluster are targets of Hh signaling in the dorsal ectoderm of Drosophila. (A) DamCi^Act (orange vertical bars) and DamCi^Rep (blue vertical bars) signal at drumstick (drm), sister of odd and bowl (sob) and odd skipped (odd). Red boxes indicate Ci motif clusters matching the motif YGSGDGGNC (see Table S1 in the supplementary material). (B-E) Wild-type expression patterns of ptc (B), drm (C), sob (D) and odd (E). Expression of ptc coincides with the anterior margin of the tracheal pit (white line in B). Stripe of drm expression lies immediately posterior to hh-lacZ (brown staining in C). (F-I) Expression of drm (G), sob (H), and odd (I) expands posteriorly by two to three cell diameters in daGal4>UAS Ci^Act. ptc expression expands similarly (F; arrow) and is activated in more cells of each parasegment. (J) Diagram of Hh target gene activation in the dorsal ectoderm. ptc expression (tan) on the anterior and posterior flanks of each hh stripe (blue) expands with ubiquitous Ci^Act (right; red stripes), but expression of Hh targets odd, drm and sob (brown), which in wild type (WT, left) is posterior to the dorsal portion of each hh stripe, expands only on the dorsal-posterior flank.

Novel Hh target genes in Drosophila embryonic visual primordia respond to Ci^Act in a tissue-specific manner. (A-I) Lateral view of the visual primordia. Lips of the optic lobe (OL) placode are outlined with dotted lines. Anterior is to the left. In the wild type, drl (A) and eya (B) are expressed in the POL. sna (C) is expressed in the most dorsal cells (arrowheads) of both the AOL and POL. In the hh mutant, POL expression of drl (D) is absent and expression of eya (E) and sna (F) is reduced. In daGal4>Ci^Act mutants, expression of drl (G), eya (H) and sna (I) is unchanged. (J-O) Dorsal view of the visual primordia. Expression of drl (J), eya (K) and sna (L) in the wild type is shown. Note the absence of expression in the dorsal head ectoderm (red dashed lines). drl (M) eya (N) and sna (O) expression in daGal4>Ci^Act fuses the normally bilateral fields of OL expression.