Suppressors and enhancers of DeltaWT overexpression in the Drosophila eye. (A–D) Adult eyes. (A′–D′) Twenty-four-hour APF retinas stained with anti-Cut antibody to detect cone cells (green). (A and A′) A wild-type eye possesses an organized array of ommatidia (A); each ommatidium has four cone cells (A′). (B and B′) A GMR>DeltaWT/+ eye is small, glossy, and rough, with disorganized ommatidia (B) and an average of 3.34 cone cells/ommatidium (B′). (C and C′) A P{XP}d04859/GMR>DeltaWT eye. P{XP}d04859 mediates overexpression of Vha68-2. Adults have a larger, less glossy eye (C) with an average of 3.5 cone cells/ommatidium (C′); P < 0.005 compared with GMR>DlWT/+. (D and D′) A P{XP}d10593/GMR>DeltaWT eye. P{XP}d10593 mediates overexpression of Hr38. Adults have a smaller eye with loss of pigmentation and more disorganized ommatidia (D) with an average of 2.89 cone cells/ommatidium (D′); P < 0.005 compared with GMR>DlWT/+.

Overexpression of Amun suppresses the GMR>DeltaWT/+ cone cell phenotype. (A–C) Twenty-four-hour APF retinas stained with anti-Cut antibody (green) to detect cone cells. (A) A GMR>DeltaWT/+ retina exhibits a disorganized array of ommatidia with an average of 3.34 cone cells/ommatidium. (B) A P{XP}d03329/+;GMR>DeltaWT/+ retina exhibits suppression of the GMR>DeltaWT/+ cone cell phenotype, increasing the average to 3.5 cone cells/ommatidium. (C) A GMR>DeltaWT/+;UAS-AmunWT/+ retina exhibits greater suppression of the GMR>DeltaWT/+ cone cell phenotype, as compared to B, increasing the average to 3.73 cone cells/ommatidium (P < 0.005). (D) The proportional representation of ommatidia with two, three, four, or five cone cells. The percentage of ommatidia with four cone cells is greater for P{XP}d03329/+;GMR>DeltaWT/+ and GMR>DeltaWT/+;UAS-AmunWT/+ retinas than for GMR>DeltaWT/+ retinas. An asterisk denotes a statistically significant difference (P < 0.005) in average cone cells per ommatidium, as compared to GMR>DeltaWT/+.

AmunRNAi effectively reduces Amun∷RFP protein expression. Third larval instar wing/notal imaginal discs. (A) A pnr-Gal4 UAS-Amun∷RFP/+ disc (pnr>Amun∷RFP). Overexpression of Amun∷RFP can be detected via RFP expression (red) in the pnr expression domain. (B) A UAS-AmunRNAi/+; pnr>Amun∷RFP/+ disc. AmunRNAi successfully reduces Amun∷RFP expression, as seen by the substantial reduction of Amun∷RFP signal within the pnr expression domain.

Amun-induced loss of notal microchaetae is due to the loss of bristle organs and sensory organ precursors in the developing notum. (A and B) Nota from 31-hr APF pupae (27°) stained with MAb22C10 to detect neurons and shaft cells. (A) A wild-type notum has organized rows of microchaeta neurons and shaft cells. (B) A pnr-Gal4 UAS-Amun∷RFP/+ notum lacks staining for neurons within the pnr expression domain (outlined by dashes), indicating that the absence of external shafts is not due to the transformation of shaft/socket cells into neurons. (C and D) Nota from 15-hr APF pupae (27°) stained for β-galactosidase activity. The neur ^A101 lacZ insertion in the neuralized gene is used to mark SOPs. (C) A neur ^A101 notum exhibits wild-type rows of microchaete SOPs. (D) A pnr-Gal4 UAS-Amun∷RFP/neur^A101 notum lacks SOPs within the pnr expression domain (outlined by dashes).

Secondary tests for confirmed modifiers of the GMR>DeltaWT/+ phenotype. (A) A wild-type adult wing. (B) Adult 34B-Gal4 UAS-DeltaΔICD/+ (34B>DeltaΔICD) wings display thickened wing veins, consistent with reduced Notch signaling. (C) Adult 34B>DeltaΔICD/+;P{XP}d11183/+ wings exhibit enhancement of the 34B>DeltaΔICD wing-vein-thickening phenotype. P{XP}d11183 disrupts karst. (D) Adult P{XP}d03329/+;34B>DeltaΔICD/+ wings exhibit suppression of the 34B>DeltaΔICD wing vein-thickening phenotype. P{XP}d03329 mediates overexpression of Amun and CG1837. (E) Adult UAS-DeltaΔICD/+;C96-Gal4/+ (C96>DeltaΔICD) wings display notches along the wing margin, typical of reduced Notch signaling. (F) Adult P{XP}d10593/UAS-DeltaΔICD;C96-Gal4/+ wings exhibit enhancement of the C96>DeltaΔICD wing notching phenotype. P{XP}d10593 mediates overexpression of Hr38. (G) Adult UAS-DeltaΔICD/+;C96-Gal4/P{XP}d07162 wings exhibit suppression of the C96>DeltaΔICD wing notching phenotype. P{XP}d07162 disrupts Cysteine string protein.

Amun contains a putative DNA glycosylase domain and localizes to the nucleus. (A) Comparison of the Amun HhH DNA glycosylase domain to other known DNA glycosylases. The sequences used were the following: MPG, H. sapiens; MutY, H. sapiens; Pdg, M. tuberculosis; AlkA, B. subtilis; OGG1, D. melanogaster; Ogg1, H. sapiens; Nth1, D. melanogaster; Nth1, H. sapiens. There is little sequence similarity among these proteins; however, they share a conserved DNA-binding motif that consists of two α-helices (purple cylinders denote the approximate locations of these helices) connected by a hairpin loop with the consensus sequence LPG(V/I)G followed by a glycine/proline-rich region (green highlight) and a catalytically active aspartic acid residue (D, red highlight). The conserved H/N residue (blue highlight), following the catalytic D residue, differentiates between monofunctional (N) and bifunctional (H) glycosylases. Blue-highlighted L, P, and V residues are part of the consensus HhH domain. Red-highlighted white G residues are completely conserved in these DNA glycosylases. (B and B′) A dpp-Gal4/UAS-Amun∷RFP third larval instar salivary gland. Amun∷RFP (red) localizes to nuclei (B), as indicated by the DNA dye Vybrant Green (B′).

Amun loss-of-function, gain-of-function, and rescue experiments demonstrate a function for Amun during sensory organ development. (A) A wild-type adult notum. There are 10 organized rows of notal microchaetae including and between the two rows containing the dorsocentral macrochaetae (aDC and pDC). (B–D) Amun∷RFP overexpression phenotypes (assessed following growth at 27°). (E–H) Loss-of-function phenotypes that result from UAS-AmunRNAi expression (27° unless otherwise noted). (B) A ptc-Gal4/+;UAS-Amun∷RFP/+ notum results in loss of aSC (arrow) and pSC macrochaetae. (C) A sr-Gal4/UAS-Amun∷RFP notum exhibits severe loss of microchaetae in stripes 2 and 3 (highlighted with square brackets). (D) A pnr-Gal4 UAS-Amun∷RFP/+ notum exhibits severe loss of microchaetae across the central notum from stripe 1 to stripe 4 (highlighted with square brackets). (E) A UAS-AmunRNAi/+;UAS-Dicer2/ sca-Gal4 notum exhibits loss of aDC (data not shown), aSC (arrow), and pSC macrochaeta shafts (data not shown). (F) A UAS-AmunRNAi/+;UAS-Dicer2/ptc-Gal4 notum exhibits loss of aDC, pDC (arrow), and aSC (arrow) macrochaeta shafts, as well as misplacement of aSC macrochaetae. (G) A UAS-AmunRNAi/+;UAS-Dicer2/+;sr-Gal4/+ notum exhibits smaller microchaetae within the sr expression domain. Inset shows a magnified view of the medial notum. (H) A UAS-AmunRNAi/+;UAS-Dicer2/+;pnr-Gal4/+ notum (25°) displays smaller microchaetae and misplaced aDC macrochaetae. Inset shows a magnified view of the medial notum. (I) A UAS-AmunRNAi/+;pnr-Gal4 UAS-Amun∷RFP/+ notum. Co-expression of Amun∷RFP and AmunRNAi rescues the Amun∷RFP-induced gain-of-function phenotype (microchaeta loss) shown in D and the AmunRNAi-induced loss-of-function phenotype (smaller microchaetae) shown in H. (J) A UAS-Achaete/+;pnr-Gal4 UAS-Amun∷RFP/+ notum. Overexpression of Achaete partially rescues the Amun∷RFP overexpression phenotype (microchaeta loss) shown in D, supporting the hypothesis that loss of microchaetae occurs as a result of reductions in Achaete function. We also note the presence of ectopic macrochaetae, which may be due to Achaete overexpression. aDC, anterior dorsocentral; pDC, posterior dorsocentral; aSC, anterior scutellar; pSC, posterior scutellar.

Amun overexpression downregulates Achaete levels in a cell-autonomous manner. (A and B) Nota from 9-hr APF pupae (27°) stained with anti-Achaete antibody (green). (A, A′, and A″) A sr-Gal/UAS-myr-mRFP notum. RFP (red) reflects expression of mRFP within the sr domain. Achaete-positive cells (arrows) mark the microchaeta proneural groups during this developmental stage. (B, B′, and B″) A sr-Gal4/UAS-Amun∷RFP notum shows a severe reduction or absence of Achaete expression (arrowheads) within the sr expression domain where Amun∷RFP is overexpressed. (C, C′, and C″) A 6- to 7-hr APF notum stained with anti-Achaete antibody (green in C and C″). Amun∷RFP is overexpressed randomly throughout the notal disc in Act5C-Gal4 clones (red). Achaete protein levels are downregulated in cells in which Amun∷RFP is overexpressed (arrowheads). Achaete-positive cells can be found directly adjacent to Amun∷RFP-positive cells (arrows) in clones in all regions of the notum.