Notch pathway activity is impaired in pyd mutant proneural clusters. (A–D) Extra sensory bristles in pyd mutants. Wild-type (WT) flies have four dorsocentral (DC) macrochaetae (A, arrowheads indicate two left DCs), and pyd mutants have greater than four DCs (B, pyd^ex147/Df(3R)p-XT103, arrowheads). DC sensory bristle precursors are detected by anti-Senseless (C and D, blue and arrowheads). Two are detected in a wild-type wing imaginal disc (C); extra Senseless-positive cells are detected in pyd mutant clones (pyd^ex147 marked by the absence of GFP; D, green). Bars, 25 µm. (E) Percentage of wild-type (w-) and pyd mutant flies with the normal complement of four DC macrochaetae (indicated pyd alleles were in trans to Df(3R)p-XT103). Subsequent experiments used the hypomorphic combination pyd^tamou/pyd^C5 in which ∼30% of pyd^tamou/pyd^C5 flies have four DC macrochaetae. n ≥ 4. (F) Percentage of flies with four DC macrochaetae in the indicated genotypes. Gray bars, control genotypes; 100% have four DC macrochaetae. Black bars, pyd mutant flies ± reduction in Notch. A pyd DC phenotype is manifested by fewer flies having four macrochaetae and is strongly enhanced by removing one copy of Notch (P < 0.0001). n = 4. (E and F) Error bars represent SD. (G and H) Expression of the Notch reporter E(spl)m8–lacZ (red) is lost in several hypomorphic pyd^tamou (G) or null pyd^ex147 (H) pyd mutant clones that cross the dorsoventral boundary (e.g., arrowheads). Confocal xy section, pyd clones marked by the absence of GFP. Bars, 10 µm.