Identification of Photoshop Mutants

<div><p>(A) FLP/MARCM system used to generate GFP-labeled homozygous mutant tracheal cell clones in heterozygous animals. Heterozygous cells have mutations (asterisk) in trans to a <i>GAL80</i> transgene (triangle) that inhibits expression of a tracheal-specific, <i>btl-GAL4</i>-driven <i>GFP</i> transgene on another chromosome (not shown). All cells also carry a heat-inducible FLP transgene on another chromosome (not shown). Following S phase of the cell cycle (S), FLP-mediated recombination (R) at the FRT, and M phase (M), homozygous mutant tracheal cells are segregated that lack the <i>GAL80</i> transgene and express GFP (green), distinguishing them from homozygous <i>GAL80</i> sister cells and nonrecombined heterozygous cells.</p><p>(B) Anteriors of L3 larvae with wild-type (WT; <i>y w</i>) control clones (top) or photoshop mutant <i>14J</i> clones (bottom). Larvae were photographed next to each other to facilitate comparison. Note strong GFP signal in <i>14J</i> clones but barely detectable signal in control clones. Bar, 0.5 mm.</p><p>(C) GFP signal is increased throughout tracheal system in photoshop mutant <i>25G</i> larvae. A wild-type <i>(y w FRT^19A/Y; btl-GAL4, UAS-GFP)</i> control larva is shown above, and a hemizygous <i>25G/Y</i> photoshop mutant <i>(25G/Y; btl-GAL4, UAS-GFP)</i> larva is shown below. Bar, 1 mm.</p><p>(D) GFP signal is increased throughout tracheal system in photoshop <i>32AP</i> larvae. Above is a control heterozygous (<i>32AP/+</i>; <i>btl-GAL4, UAS-GFP</i>/+) larva showing weak GFP signal throughout tracheal system. In the middle is a <i>32AP/Df</i> mutant female (<i>32AP/Df; btl-GAL4, UAS-GFP</i>/+) larva showing enhanced GFP signal throughout. Below is a <i>32AP/Y</i> mutant male (<i>32AP</i>/Y<i>; btl-GAL4, UAS-GFP</i>/+) larva: GFP signal is indistinguishable from that observed in a <i>32AP/Df</i> larva, indicating <i>32AP</i> is an amorphic (null) allele. Bar, 1 mm.</p><p>(E) Effect of a photoshop mutation on a <i>DsRed</i> transgene. Photoshop <i>14J</i> tracheal clone in <i>y w 14J FRT^19A/ w FRT^19A, FLP122</i> larva carrying <i>btl-GAL4</i> and <i>UAS-DsRed1</i> transgenes. Two unicellular tracheal tubes are indicated by brackets; <i>14J</i>⁻ clone has higher DsRed1 signal than neighboring wild-type (<i>14J⁺</i>) cell. Bar, 25 μm.</p><p>(F) Effect of photoshop <i>25G</i> mutation outside the tracheal system. A mixture of wild-type and <i>25G/Y</i> mutant animals carrying <i>da-GAL4, UAS-CD8:GFP</i> is shown. GFP signal is increased throughout all tissues of the <i>25G</i> mutants. <i>25G</i> genotype of high-signal larvae was confirmed by PCR. Bar, 2 mm.</p><p>(G) Effect of photoshop <i>32AP</i> mutation in epidermis. A wild-type larva is shown above, and below is a <i>32AP/Y</i> mutant larva (bottom) carrying <i>e22c-GAL4, UAS-nls:DsRed2,</i> which expresses a nuclear localized DsRed2 in epidermis and other epithelial tissues. Animals were photographed next to each other. The nls:DsRed2 signal is enhanced in the epidermis of the <i>32AP/Y</i> mutant. Bar, 1 mm.</p><p>(H) Effect of photoshop mutants on <i>GFP</i> RNA levels. Quantitative real time RT-PCR was done on the <i>GFP</i> transcript in wild-type <i>(y w FRT^19A/Y), 25G/Y,</i> and <i>32AP/Y</i> L3 larvae carrying one copy of <i>da-GAL4</i> and <i>UAS-CD8:GFP</i>. Results of replicate reactions were individually normalized to parallel reactions on <i>rp18LA</i> transcript. Values shown are mean ± 2 standard errors of the mean normalized to value in wild type.</p>