Crossing scheme for the screen. EMS-mutagenized males were crossed to females with the C765-Gal4,UAS-Smo5A tester chromosome (C5). The F₁ progeny with enhancement or suppression of the C5 phenotype were crossed to the backcross strain (containing the C5 chromosome, the second chromosome dominant marker Bristle, and second and third chromosome balancers). F₂ flies that showed penetrant modification of the C5 phenotype were again crossed to the backcross strain to generate a balanced mutant stock and to map the modifying mutation to the second or third chromosome.

Modification of C765-Gal4,UAS-Smo5A by third chromosome complementation groups. The 12 complementation groups on the third chromosome enhance the C5 phenotype (see Figure 1E for comparison). The group and mutant number are indicated.

Group C is required for Hedgehog signaling. Confocal images of third instar wing imaginal discs containing clones of cells mutant for group C (loss of green GFP expression in A, B, D, E, and H) and stained for full-length Ci protein (red in A, C, D, and F) or anti-β-Gal (red in G–I) to show expression of dpp-LacZ. In wild-type tissue, Ci-155 is stabilized by Hh signaling, and this results in a broad band of more heavily stained cells on the posterior side of the A/P boundary. Clones of cells mutant for group C fail to upregulate Ci-155 (A and C) except, notably, those cells that are immediately adjacent to the Hh source. Also, wild-type cells on the opposite side of the clone from the source of Hh nonautonomously fail to upregulate Ci-155 (arrow in C). Ci-155 is upregulated normally in anterior cells (arrow in F) when these cells are adjacent to a large, posterior clone at the A/P boundary (loss of green GFP expression), suggesting that group C in not required in Hh-producing cells. In wild-type wing imaginal discs, dpp-lacZ is expressed in a stripe along the posterior side of the A/P boundary (red in G). In clones of cells for group C (loss of green GFP expression in H), dpp-lacZ expression (red in H and I) was lost, except in a single row of cells immediately adjacent to the A/P boundary and the Hh source.

Smo5A is a dominant negative. (A) The primary sequence of smoothened, signal sequence (gray), transmembrane domains (green) C-terminal cytoplasmic tail (blue), and five putative PKA phosphorylation sites (red) that were mutated to alanines are highlighted. Expression of Smo5A in flip-out clones (green GFP-expressing cells in B) represses the expression of Patched expression (red; B and C). Expression of Smo5A in the wing with the C765-Gal4 (E) driver causes a reduction of the central portion of the wing between longitudinal veins III and IV and fusion of these veins proximal to the anterior cross-vein (compare to wild type; D). These phenotypes are strongly enhanced in flies heterozygous for smo (F) and are completely suppressed by removing one copy of ptc. Longitudinal veins I–V and the anterior cross-vein (ACV) are indicated in D.

Summary of mapping complementation groups on the third chromosome. The cytological map positions or intervals in the third chromosome complementation groups are indicated by bars and open boxes, respectively.

Group D mutations are alleles of pavarotti. (A) The cytological positions, and the distance in centimorgans from group D, of the insertions used for P-element-mediated meiotic recombination mapping are indicated on the top line. The region around EP(3)1135, which is only 0.015 cM from group D, is shown in the bottom line. pav is ∼10 kb from the EP(3)1135 insertion site. Mutations in pav modify the C5 phenotype. Two alleles of pav identified in the screen, pav⁸³¹ and pav²⁰⁴⁶, enhance the C5 phenotype (B and D, respectively) whereas the third allele, pav⁹⁶³, suppresses the C5 phenotype (C). The previously described pav^B200 allele also enhances the C5 phenotype (E).

Wg and Dpp signaling are disrupted in jaft mutants. Dpp is expressed along the A/P compartment boundary and signals in a graded manner in both anterior and posterior compartments. Pathway activation can be detected with antibodies against the phosphorylated form of the downstream component Mad (P-Mad, red in A and C). In clones of cells mutant for jaft (absence of green GFP expression in A and B), there is no detectable phosphorylated Mad (arrows in B and C). Wg secreted from the D/V margin activated the graded expression of the Dll target gene in both dorsal and ventral compartments (red in D and F). In clones of cells mutant for jaft (absence of green GFP expression in D and E), only those cells closest to the Wg source express Dll, and in jaft mutant clones, Dll expression ends in a sharp border rather than the graded expression in wild-type tissue.